Human RNase H1 oligonucleotide compositions thereof

ABSTRACT

The present invention provides oligonucleotides that can serve as substrates for human Type 2 RNase H. The present invention is also directed to methods of using these oligonucleotides in enhancing antisense oligonucleotide therapies.

CROSS REFERENCE

This application is a continuation of U.S. patent application Ser. No.10/616,009, filed Jul. 8, 2003, now pending, which is a continuation ofU.S. patent application Ser. No. 09/409,926, filed Sep. 30, 1999, nowU.S. Pat. No. 6,617,442, both of which are hereby incorporated byreference in their entirety.

FIELD OF THE INVENTION

The present invention relates to a human Type 2 RNase H which has nowbeen cloned, expressed and purified to electrophoretic homogeneity. Thepresent invention further relates to oligonucleotide compositions thatmay serve as substrates for human RNase H1 or human Type 2 RNase H.

BACKGROUND OF THE INVENTION

RNase H hydrolyzes RNA in RNA-DNA hybrids. This enzyme was firstidentified in calf thymus but has subsequently been described in avariety of organisms (Stein, H. and Hausen, P., Science, 1969, 166,393-395; Hausen, P. and Stein, H.,Eur. J. Biochem., 1970, 14, 278-283).RNase H activity appears to be ubiquitous in eukaryotes and bacteria(Itaya, M. and Kondo K. Nucleic Acids Res., 1991, 19, 4443-4449; Itayaet al., Mol. Gen. Genet., 1991 227, 438-445; Kanaya, S., and Itaya, M.,J. Biol. Chem., 1992, 267, 10184-10192; Busen, W., J. Biol. Chem., 1980,255, 9434-9443; Rong, Y. W. and Carl, P. L., 1990, Biochemistry 29,383-389; Eder et al., Biochimie, 1993 75, 123-126). Although RNase Hsconstitute a family of proteins of varying molecular weight, nucleolyticactivity and substrate requirements appear to be similar for the variousisotypes. For example, all RNase Hs studied to date function asendonucleases, exhibiting limited sequence specificity and requiringdivalent cations (e.g., Mg²⁺, Mn²⁺) to produce cleavage products with 5′phosphate and 3′ hydroxyl termini (Crouch, R. J., and Dirksen, M. L.,Nuclease, Linn, S, M., & Roberts, R. J., Eds., Cold Spring HarborLaboratory Press, Plainview, N.Y. 1982, 211-241).

In addition to playing a natural role in DNA replication, RNase H hasalso been shown to be capable of cleaving the RNA component of certainoligonucleotide-RNA duplexes. While many mechanisms have been proposedfor oligonucleotide mediated destabilization of target RNAs, the primarymechanism by which antisense oligonucleotides are believed to cause areduction in target RNA levels is through this RNase H action. Monia etal., J. Biol. Chem., 1993, 266:13, 14514-14522. In vitro assays havedemonstrated that oligonucleotides that are not substrates for RNase Hcan inhibit protein translation (Blake et al., Biochemistry, 1985, 24,6139-4145) and that oligonucleotides inhibit protein translation inrabbit reticulocyte extracts that exhibit low RNase H activity. However,more efficient inhibition was found in systems that supported RNase Hactivity (Walder, R. Y. and Walder, J. A., Proc. Nat'l Acad. Sci. USA,1988, 85, 5011-5015; Gagnor et al., Nucleic Acid Res., 1987, 15,10419-10436; Cazenave et al., Nucleic Acid Res., 1989, 17, 4255-4273;and Dash et al., Proc. Nat'l Acad. Sci. USA, 1987, 84, 7896-7900.

Oligonucleotides commonly described as “antisense oligonucleotides”comprise nucleotide sequences sufficient in identity and number toeffect specific hybridization with a particular nucleic acid. Thisnucleic acid or the protein(s) it encodes is generally referred to asthe “target.” Oligonucleotides are generally designed to bind eitherdirectly to mRNA transcribed from, or to a selected DNA portion of, apreselected gene target, thereby modulating the amount of proteintranslated from the mRNA or the amount of mRNA transcribed from thegene, respectively. Antisense oligonucleotides may be used as researchtools, diagnostic aids, and therapeutic agents.

“Targeting” an oligonucleotide to the associated nucleic acid, in thecontext of this invention, also refers to a multistep process whichusually begins with the identification of the nucleic acid sequencewhose function is to be modulated. This may be, for example, a cellulargene (or mRNA transcribed from the gene) whose expression is associatedwith a particular disorder or disease state, or a foreign nucleic acidfrom an infectious agent. The targeting process also includesdetermination of a site or sites within this gene for theoligonucleotide interaction to occur such that the desired effect,either detection or modulation of expression of the protein, willresult.

RNase H1 from E.coli is the best-characterized member of the RNase Hfamily. The 3-dimensional structure of E.coli RNase HI has beendetermined by x-ray crystallography, and the key amino acids involved inbinding and catalysis have been identified by site-directed mutagenesis(Nakamura et al., Proc. Natl. Acad. Sci. USA, 1991, 88, 11535-11539;Katayanagi et al., Nature, 1990, 347, 306-309; Yang et al., Science,1990, 249, 1398-1405; Kanaya et al., J. Biol. Chem., 1991, 266,11621-11627). The enzyme has two distinct structural domains. The majordomain consists of four helices and one large sheet composed of threeantiparallel strands. The Mg²⁺ binding site is located on the sheet andconsists of three amino acids, Asp-10, Glu-48, and Gly-11 (Katayanagi etal., Proteins: Struct., Funct., Genet., 1993, 17, 337-346). Thisstructural motif of the Mg²⁺ binding site surrounded by strands issimilar to that in DNase 3 (Suck, D., and Oefner, C., Nature, 1986, 321,620-625). The minor domain is believed to constitute the predominantbinding region of the enzyme and is composed of an helix terminatingwith a loop. The loop region is composed of a cluster of positivelycharged amino acids that are believed to bind electrostatistically tothe minor groove of the DNA/RNA heteroduplex substrate. Although theconformation of the RNA/DNA substrate can vary, from A-form to B-formdepending on the sequence composition, in general RNA/DNA heteroduplexesadopt an A-like geometry (Pardi et al., Biochemistry, 1981, 20,3986-3996; Hall, K. B., and Mclaughlin, L. W., Biochemistry, 1991, 30,10606-10613; Lane et al., Eur. J. Biochem., 1993, 215, 297-306). Theentire binding interaction appears to comprise a single helical turn ofthe substrate duplex. Recently the binding characteristics, substraterequirements, cleavage products and effects of various chemicalmodifications of the substrates on the kinetic characteristics of E.coliRNase HI have been studied in more detail (Crooke, S. T. et al.,Biochem. J., 1995, 312, 599-608; Lima, W. F. and Crooke, S. T.,Biochemistry, 1997, 36, 390-398; Lima, W. F. et al., J. Biol. Chem.,1997, 272, 18191-18199; Tidd, D. M. and Worenius, H. M., Br. J. Cancer,1989, 60, 343; Tidd, D. M. et al., Anti-Cancer Drug Des., 1988, 3, 117.

In addition to RNase HI, a second E.coli RNase H, RNase HII has beencloned and characterized (Itaya, M., Proc. Natl. Acad. Sci. USA, 1990,87, 8587-8591). It is comprised of 213 amino acids while RNase HI is 155amino acids long. E. coli RNase HIM displays only 17% homology withE.coli RNase HI. An RNase H cloned from S. typhimurium differed fromE.coli RNase HI in only 11 positions and was 155 amino acids in length(Itaya, M. and Kondo K., Nucleic Acids Res., 1991, 19, 4443-4449; Itayaet al., Mol. Gen. Genet., 1991, 227, 438-445). An enzyme cloned from S.cerevisae was 30% homologous to E.coli RNase HI (Itaya, M. and Kondo K.,Nucleic Acids Res., 1991, 19,4443-4449; Itaya et al., Mol. Gen. Genet.,1991, 227, 438-445). Thus, to date, no enzyme cloned from a speciesother than E. coli has displayed substantial homology to E.coli RNase HII.

Proteins that display RNase H activity have also been cloned andpurified from a number of viruses, other bacteria and yeast(Wintersberger, U. Pharmac. Ther., 1990, 48, 259-280). In many cases,proteins with RNase H activity appear to be fusion proteins in whichRNase H is fused to the amino or carboxy end of another enzyme, often aDNA or RNA polymerase. The RNase H domain has been consistently found tobe highly homologous to E.coli RNase HI, but because the other domainsvary substantially, the molecular weights and other characteristics ofthe fusion proteins vary widely.

In higher eukaryotes two classes of RNase H have been defined based ondifferences in molecular weight, effects of divalent cations,sensitivity to sulfhydryl agents and immunological cross-reactivity(Busen et al., Eur. J. Biochem., 1977, 74, 203-208). RNase H Type 1enzymes are reported to have molecular weights in the 68-90 kDa range,be activated by either Mn²⁺ or Mg²⁺ and be insensitive to sulfhydrylagents. In contrast, RNase H Type 2 enzymes have been reported to havemolecular weights ranging from 31-45 kDa, to require Mg²⁺ to be highlysensitive to sulfhydryl agents and to be inhibited by Mn²⁺ (Busen, W.,and Hausen, P., Eur. J. Biochem., 1975, 52, 179-190; Kane, C. M.,Biochemistry, 1988, 27, 3187-3196; Busen, W., J. Biol. Chem., 1982, 257,7106-7108.).

An enzyme with Type 2 RNase H characteristics has been purified to nearhomogeneity from human placenta (Frank et al., Nucleic Acids Res., 1994,22, 5247-5254). This protein has a molecular weight of approximately 33kDa and is active in a pH range of 6.5-10, with a pH optimum of 8.5-9.The enzyme requires Mg²⁺ and is inhibited by Mn²⁺ and n-ethyl maleimide.The products of cleavage reactions have 3′ hydroxyl and 5′ phosphatetermini.

Despite the substantial information about members of the RNase familyand the cloning of a number of viral, prokaryotic and yeast genes withRNase H activity, until now, no mammalian RNase H had been cloned. Thishas hampered efforts to understand the structure of the enzyme(s), theirdistribution and the functions they may serve.

In the present invention, a cDNA of human RNase H with Type 2characteristics and the protein expressed thereby are provided.

SUMMARY OF THE INVENTION

The present invention provides oligonucleotides that can serve assubstrates for human RNase H1. These oligonucleotides are mixed sequenceoligonucleotides comprising at least two portions wherein a firstportion is capable of supporting human RNase H1 cleavage of acomplementary target RNA and a further portion which is not capable ofsupporting such human RNase H1 cleavage.

The present invention provides a mixed sequence oligonucleotidecomprising at least 12 nucleotides and having a 3′ end and a 5′ end,said oligonucleotide being divided into a first portion and a furtherportion,

said first portion being capable of supporting cleavage of acomplementary target RNA by human RNase H1 polypeptide,

said further portion being incapable of supporting said RNase Hcleavage;

wherein said first portion comprises at least 6 nucleotides and ispositioned in said oligonucleotide such that at least one of said 6nucleotides is 8 to 12 nucleotides from the 3′ end of saidoligonucleotide.

In a preferred embodiment the oligonucleotide comprises at least one CAnucleotide sequence. In another embodiment the first portion of themixed sequence oligonucleotide of the present invention comprisesnucleotides having a B-form conformational geometry. In a furtherembodiment each of the nucleotides of the first portion of theoligonucleotide are 2′-deoxyribonucleotides. In a still furtherembodiment each of the nucleotides of the first portion of theoligonucleotide is a 2′-F arabinonucleotide or a 2′-OHarabinonucleotide. In yet another embodiment the nucleotides of thefirst portion are joined together in a continuous sequence by phosphate,phosphorothioate, phosphorodithioate or boranophosphate linkages. In yeta further embodiment all of the nucleotides of the further portion ofthe oligonucleotide are joined together in a continuous sequence by3′-5′ phosphodiester, 2′-5′ phosphodiester, phosphorothioate, Spphosphorothioate, Rp phosphorothioate, phosphorodithioate,3′-deoxy-3′-amino phosphoroamidate, 3′-methylenephosphonate,methylene(methylimino), dimethylhydrazino, amide 3, amide 4 orboranophosphate linkages.

Yet another object of the present invention is to provide methods foridentifying agents which modulate activity and/or levels of human RNaseH1. In accordance with this aspect, the polynucleotides and polypeptidesof the present invention are useful for research, biological andclinical purposes. For example, the polynucleotides and polypeptides areuseful in defining the interaction of human RNase H1 and antisenseoligonucleotides and identifying means for enhancing this interaction sothat antisense oligonucleotides are more effective at inhibiting theirtarget mRNA.

Yet another object of the present invention is to provide a method ofprognosticating efficacy of antisense therapy of a selected diseasewhich comprises measuring the level or activity of human RNase H in atarget cell of the antisense therapy. Similarly, oligonucleotides can bescreened to identify those oligonucleotides which are effectiveantisense agents by measuring binding of the oligonucleotide to thehuman RNase H1.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the effects of conditions on human RNase H1 activity.

FIG. 2 shows a denaturing polyacrylamide gel analysis of human RNase H1cleavage of a 17-mer RNA-DNA gapmer duplex.

FIG. 3 shows analysis of human Rnase H1 cleavage of a 25-mer Ras RNAhybridized with phosphodiester oligodeoxynucleotides of differentlengths.

FIG. 4 shows analysis of human RNase H1 cleavage of RNA-DNA duplexeswith different sequences, length and 3′ or 5′ overhangs.

FIG. 5 shows product and processivity analysis of human RNase H1cleavage on 17-mer Ras RNA-DNA duplexes.

FIG. 6 provides the human Type 2 RNase H primary sequence (286 aminoacids; SEQ ID NO: 1) and sequence comparisons with chicken (293 aminoacids; SEQ ID NO: 2), yeast (348 amino acids; SEQ ID NO: 3) and E. coliRNase H1 (155 amino acids; SEQ ID NO: 4) as well as an EST deduced mouseRNase H homolog (GenBank accession no. AA389926 and AA518920; SEQ ID NO:5). Boldface type indicates amino acid residues identical to human. “@”indicates the conserved amino acid residues implicated in E. coli RNaseH1 Mg²⁺ binding site and catalytic center (Asp-10, Gly-11, Glu-48 andAsp-70). “*” indicates the conserved residues implicated in E. coliRNases H1 for substrate binding.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

A Type 2 human RNase H has now been cloned and expressed. The enzymeencoded by this cDNA is inactive against single-stranded RNA,single-stranded DNA and double-stranded DNA. However, this enzymecleaves the RNA in an RNA/DNA duplex and cleaves the RNA in a duplexcomprised of RNA and a chimeric oligonucleotide with 2′ methoxy flanksand a 5-deoxynucleotide center gap. The rate of cleavage of the RNAduplexed with this so-called “deoxy gapmer” was significantly slowerthan observed with the full RNA/DNA duplex. These properties areconsistent with those reported for E.coli RNase H1 (Crooke et al.,Biochem. J., 1995, 312, 599-608; Lima, W. F. and Crooke, S. T.,Biochemistry, 1997, 36, 390-398). They are also consistent with theproperties of a human Type 2 RNase H protein purified from placenta, asthe molecular weight (32 kDa) is similar to that reported by Frank etal., Nucleic Acids Res., 1994, 22, 5247-5254) and the enzyme isinhibited by Mn²⁺.

Thus, in accordance with one aspect of the present invention, there areprovided isolated polynucleotides which encode human Type 2 RNase Hpolypeptides having the deduced amino acid sequence of FIG. 1. By“polynucleotides” it is meant to include any form of RNA or DNA such asmRNA or cDNA or genomic DNA, respectively, obtained by cloning orproduced synthetically by well known chemical techniques. DNA may bedouble- or single-stranded. Single-stranded DNA may comprise the codingor sense strand or the non-coding or antisense strand.

Methods of isolating a polynucleotide of the present invention viacloning techniques are well known. For example, to obtain the cDNAcontained in ATCC Deposit No. 98536, primers based on a search of theXREF database were used. An approximately 1 Kb cDNA corresponding to thecarboxy terminal portion of the protein was cloned by 3′ RACE. Sevenpositive clones were isolated by screening a liver cDNA library withthis 1 Kb cDNA. The two longest clones were 1698 and 1168 base pairs.They share the same 5′ untranslated region and protein coding sequencebut differ in the length of the 3′ UTR. A single reading frame encodinga 286 amino acid protein (calculated mass: 32029.04 Da) was identified(FIG. 1). The proposed initiation codon is in agreement with themammalian translation initiation consensus sequence described by Kozak,M., J. Cell Biol., 1989, 108, 229-241, and is preceded by an in-framestop codon. Efforts to clone cDNA's with longer 5′ UTR's from both humanliver and lymphocyte cDNA's by 5′ RACE failed, indicating that the1698-base-pair clone was full length.

In a preferred embodiment, the polynucleotide of the present inventioncomprises the nucleic acid sequence of the cDNA contained within ATCCDeposit No. 98536. The deposit of E. coli DH5 containing a BLUESCRIPT®plasmid containing a human Type 2 RNase H cDNA was made with theAmerican Type Culture Collection, 12301 Park Lawn Drive, Rockville, Md.20852, USA, on Sep. 4, 1997 and assigned ATCC Deposit No. 98536. Thedeposited material is a culture of E. coli DH5 containing a BLUESCRIPT®plasmid (Stratagene, La Jolla Calif.) that contains the full-lengthhuman Type 2 RNase H cDNA. The deposit has been made under the terms ofthe Budapest Treaty on the international recognition of the deposit ofmicro-organisms for the purposes of patent procedure. The culture willbe released to the public, irrevocably and without restriction to thepublic upon issuance of this patent. The sequence of the polynucleotidecontained in the deposited material and the amino acid sequence of thepolypeptide encoded thereby are controlling in the event of any conflictwith the sequences provided herein. However, as will be obvious to thoseof skill in the art upon this disclosure, due to the degeneracy of thegenetic code, polynucleotides of the present invention may compriseother nucleic acid sequences encoding the polypeptide of FIG. 1 andderivatives, variants or active fragments thereof.

Another aspect of the present invention relates to the polypeptidesencoded by the polynucleotides of the present invention. In a preferredembodiment, a polypeptide of the present invention comprises the deducedamino acid sequence of human Type 2 RNase H provided in FIG. 1 as SEQ IDNO: 1. However, by “polypeptide” it is also meant to include fragments,derivatives and analogs of SEQ ID NO: 1 which retain essentially thesame biological activity and/or function as human Type 2 RNase H.Alternatively, polypeptides of the present invention may retain theirability to bind to an antisense-RNA duplex even though they do notfunction as active RNase H enzymes in other capacities. In anotherembodiment, polypeptides of the present invention may retain nucleaseactivity but without specificity for the RNA portion of an RNA/DNAduplex. Polypeptides of the present invention include recombinantpolypeptides, isolated natural polypeptides and synthetic polypeptides,and fragments thereof which retain one or more of the activitiesdescribed above.

In a preferred embodiment, the polypeptide is prepared recombinantly,most preferably from the culture of E. coli of ATCC Deposit No. 98536.Recombinant human RNase H fused to histidine codons (his-tag; in thepresent embodiment six histidine codons were used) expressed in E.colican be conveniently purified to electrophoretic homogeneity bychromatography with Ni-NTA followed by C4 reverse phase HPLC. Thepurified recombinant polypeptide of SEQ ID NO: 1 is highly homologous toE.coli RNase H, displaying nearly 34% amino acid identity with E.coliRNase H1. FIG. 1 compares the protein sequences deduced from human RNaseH cDNA (SEQ ID NO: 1) with those of chicken (SEQ ID NO: 2), yeast (SEQID NO: 3) and E.coli RNase HI (Gene Bank accession no. 1786408; SEQ IDNO: 4), as well as an EST deduced mouse RNase H homolog (Gene Bankaccession no. AA389926 and AA518920; SEQ ID NO: 5). The deduced aminoacid sequence of human RNase H (SEQ ID NO: 1) displays strong homologywith yeast (21.8% amino acid identity), chicken (59%), E.coli RNase HI(33.6%) and the mouse EST homolog (84.3%). They are all small proteins(<40 KDa) and their estimated pls are all 8.7 and greater. Further, theamino acid residues in E.coli RNase HI thought to be involved in theMg²⁺ binding site, catalytic center and substrate binding region arecompletely conserved in the cloned human RNase H sequence (FIG. 1).

The human Type 2 RNase H of SEQ ID NO: 1 is expressed ubiquitously.Northern blot analysis demonstrated that the transcript was abundant inall tissues and cell lines except the MCR-5 line. Northern blot analysisof total RNA from human cell lines and Poly A containing RNA from humantissues using the 1.7 kb full length probe or a 332-nucleotide probethat contained the 5′ UTR and coding region of human RNase H cDNArevealed two strongly positive bands with approximately 1.2 and 5.5 kbin length and two less intense bands approximately 1.7 and 4.0 kb inlength in most cell lines and tissues. Analysis with the 332-nucleotideprobe showed that the 5.5 kb band contained the 5′ UTR and a portion ofthe coding region, which suggests that this band represents apre-processed or partially processed transcript, or possibly analternatively spliced transcript. Intermediate sized bands may representprocessing intermediates. The 1.2 kb band represents the full lengthtranscripts. The longer transcripts may be processing intermediates oralternatively spliced transcripts.

RNase H is expressed in most cell lines tested; only MRC5, a breastcancer cell line, displayed very low levels of RNase H. However, avariety of other malignant cell lines including those of bladder (T24),breast (T-47D, HS578T), lung (A549), prostate (LNCap, DU145), andmyeloid lineage (HL-60), as well as normal endothelial cells (HUVEC),expressed RNase H. Further, all normal human tissues tested expressedRNase H. Again, larger transcripts were present as well as the 1.2 kbtranscript that appears to be the mature mRNA for RNase H. Normalizationbased on G3PDH levels showed that expression was relatively consistentin all of the tissues tested.

The Southern blot analysis of EcoRI digested human and various mammalianvertebrate and yeast genomic DNAs probed with the 1.7 kb probe showsthat four EcoRI digestion products of human genomic DNA (2.4, 4.6, 6.0,8.0 Kb) hybridized with the 1.7 kb probe. The blot re-probed with a 430nucleotide probe corresponding to the C-terminal portion of the proteinshowed only one 4.6 kbp EcoRI digestion product hybridized. These dataindicate that there is only one gene copy for RNase H and that the sizeof the gene is more than 10 kb. Both the full length and the shorterprobe strongly hybridized to one EcoRI digestion product of yeastgenomic DNA (about 5 kb in size), indicating a high degree ofconservation. These probes also hybridized to the digestion product frommonkey, but none of the other tested mammalian genomic DNAs includingthe mouse which is highly homologous to the human RNase H sequence.

A recombinant human RNase H (his-tag fusion protein) polypeptide of thepresent invention was expressed in E.coli and purified by Ni-NTA agarosebeads followed by C4 reverse phase column chromatography. A 36 kDaprotein copurified with activity measured after renaturation. Thepresence of the his-tag was confirmed by Western blot analyses with ananti-penta-histidine antibody (Qiagen, Germany).

Renatured recombinant human RNase H displayed RNase H activity.Incubation of 10 ng purified renatured RNase H with RNA/DNA substratefor 2 hours resulted in cleavage of 40% of the substrate. The enzymealso cleaved RNA in an oligonucleotide/RNA duplex in which theoligonucleotide was a gapmer with a 5-deoxynucleotide gap, but at a muchslower rate than the full RNA/DNA substrate. This is consistent withobservations with E.coli RNase HI (Lima, W. F. and Crooke, S. T.,Biochemistry, 1997, 36, 390-398). It was inactive againstsingle-stranded RNA or double-stranded RNA substrates and was inhibitedby Mn²⁺. The molecular weight (˜36kDa) and inhibition by Mn²⁺ indicatethat the cloned enzyme is highly homologous to E.coli RNase HI and hasproperties consistent with those assigned to Type 2 human RNase H.

The sites of cleavage in the RNA in the full RNA/DNA substrate and thegapmer/RNA duplexes (in which the oligonucleotide gapmer had a5-deoxynucleotide gap) resulting from the recombinant enzyme weredetermined. In the full RNA/DNA duplex, the principal site of cleavagewas near the middle of the substrate, with evidence of less prominentcleavage sites 3′ to the primary cleavage site. The primary cleavagesite for the gapmer/RNA duplex was located across the nucleotideadjacent to the junction of the 2′ methoxy wing and oligodeoxynucleotide gap nearest the 3′ end of the RNA. Thus, the enzyme resultedin a major cleavage site in the center of the RNA/DNA substrate and lessprominent cleavages to the 3′ side of the major cleavage site. The shiftof its major cleavage site to the nucleotide in apposition to the DNA 2′methoxy junction of the 2′ methoxy wing at the 5′ end of the chimericoligonucleotide is consistent with the observations for E.coli RNase HI(Crooke et al. (1995) Biochem. J. 312, 599-608; Lima, W. F. and Crooke,S. T. (1997) Biochemistry 36, 390-398). The fact that the enzyme cleavesat a single site in a 5-deoxy gap duplex indicates that the enzyme has acatalytic region of similar dimensions to that of E.coli RNase HI.

Accordingly, expression of large quantities of a purified human RNase Hpolypeptide of the present invention is useful in characterizing theactivities of a mammalian form of this enzyme. In addition, thepolynucleotides and polypeptides of the present invention provide ameans for identifying agents which enhance the function of antisenseoligonucleotides in human cells and tissues.

For example, a host cell can be genetically engineered to incorporatepolynucleotides and express polypeptides of the present invention.Polynucleotides can be introduced into a host cell using any number ofwell known techniques such as infection, transduction, transfection ortransformation. The polynucleotide can be introduced alone or inconjunction with a second polynucleotide encoding a selectable marker.In a preferred embodiment, the host comprises a mammalian cell. Suchhost cells can then be used not only for production of human Type 2RNase H, but also to identify agents which increase or decrease levelsof expression or activity of human Type 2 RNase H in the cell. In theseassays, the host cell would be exposed to an agent suspected of alteringlevels of expression or activity of human Type 2 RNase in the cells. Thelevel or activity of human Type 2 RNase in the cell would then bedetermined in the presence and absence of the agent. Assays to determinelevels of protein in a cell are well known to those of skill in the artand include, but are not limited to, radioimmunoassays, competitivebinding assays, Western blot analysis and enzyme linked immunosorbentassays (ELISAs). Methods of determining increase activity of the enzyme,and in particular increased cleavage of an antisense-mRNA duplex can beperformed in accordance with the teachings of Example 5. Agentsidentified as inducers of the level or activity of this enzyme may beuseful in enhancing the efficacy of antisense oligonucleotide therapies.

The present invention also relates to prognostic assays wherein levelsof RNase in a cell type can be used in predicting the efficacy ofantisense oligonucleotide therapy in specific target cells. High levelsof RNase in a selected cell type are expected to correlate with higherefficacy as compared to lower amounts of RNase in a selected cell typewhich may result in poor cleavage of the mRNA upon binding with theantisense oligonucleotide. For example, the MRC5 breast cancer cell linedisplayed very low levels of RNase H as compared to other malignant celltypes. Accordingly, in this cell type it may be desired to use antisensecompounds which do not depend on RNase H activity for their efficacy.Similarly, oligonucleotides can be screened to identify those which areeffective antisense agents by contacting human Type 2 RNase H with anoligonucleotide and measuring binding of the oligonucleotide to thehuman Type 2 RNase H. Methods of determining binding of two moleculesare well known in the art. For example, in one embodiment, theoligonucleotide can be radiolabeled and binding of the oligonucleotideto human Type 2 RNase H can be determined by autoradiography.Alternatively, fusion proteins of human Type 2 RNase H withglutathione-S-transferase or small peptide tags can be prepared andimmobilized to a solid phase such as beads. Labeled or unlabeledoligonucleotides to be screened for binding to this enzyme can then beincubated with the solid phase. Oligonucleotides which bind to theenzyme immobilized to the solid phase can then be identified either bydetection of bound label or by eluting specifically the boundoligonucleotide from the solid phase. Another method involves screeningof oligonucleotide libraries for binding partners. Recombinant tagged orlabeled human Type 2 RNase H is used to select oligonucleotides from thelibrary which interact with the enzyme. Sequencing of theoligonucleotides leads to identification of those oligonucleotides whichwill be more effective as antisense agents.

The oligonucleotides of the present invention are formed from aplurality of nucleotides that are joined together via internucleotidelinkages. While joined together as a unit in the oligonucleotide, theindividual nucleotides of oligonucleotides are of several types. Each ofthese types contribute unique properties to the oligonucleotide. A firsttype of nucleotides are joined together in a continuous sequence thatforms a first portion of the oligonucleotide. The remaining nucleotidesare of at least one further type and are located in one or moreremaining portions or locations within the oligonucleotide. Thus, theoligonucleotides of the invention include a nucleotide portion thatcontributes one set of attributes and a further portion (or portions)that contributes another set of attributes.

One attribute that is desirable is eliciting RNase H activity. To elicitRNase H activity, a portion of the oligonucleotides of the invention isselected to have B-form like conformational geometry. The nucleotidesfor this B-form portion are selected to specifically includeribo-pentofuranosyl and arabino-pentofuranosyl nucleotides.2′-Deoxy-erythro-pentfuranosyl nucleotides also have B-form geometry andelicit RNase H activity. While not specifically excluded, if2′-deoxy-erythro-pentfuranosyl nucleotides are included in the B-formportion of an oligonucleotide of the invention, such2′-deoxy-erythro-pentfuranosyl nucleotides preferably does notconstitute the totality of the nucleotides of that B-form portion of theoligonucleotide, but should be used in conjunction with ribonucleotidesor an arabino nucleotides. As used herein, B-form geometry is inclusiveof both C2′-endo and O4′-endo pucker, and the ribo and arabinonucleotides selected for inclusion in the oligonucleotide B-form portionare selected to be those nucleotides having C2′-endo conformation orthose nucleotides having O4′-endo conformation. This is consistent withBerger, et. al., Nucleic Acids Research, 1998, 26, 2473-2480, whopointed out that in considering the furanose conformations in whichnucleosides and nucleotides reside, B-form consideration should also begiven to a O4′-endo pucker contribution.

A-form nucleotides are nucleotides that exhibit C3′-endo pucker, alsoknown as north, or northern, pucker. In addition to the B-formnucleotides noted above, the A-form nucleotides can be C3′-endo puckernucleotides or can be nucleotides that are located at the 3′ terminus,at the 5′ terminus, or at both the 3′ and the 5′ terminus of theoligonucleotide. Alternatively, A-form nucleotides can exist both in aC3′-endo pucker and be located at the ends, or termini, of theoligonucleotide. In selecting nucleotides that have C3′-endo pucker orin selecting nucleotides to reside at the 3′ or 5′ ends of theoligonucleotide, consideration is given to binding affinity and nucleaseresistance properties that such nucleotides need to impart to theresulting the oligonucleotide.

Nucleotides selected to reside at the 3′ or 5′ termini ofoligonucleotides of the invention are selected to impart nucleaseresistance to the oligonucleotide. This nuclease resistance can also beachieved via several mechanisms, including modifications of the sugarportions of the nucleotide units of the oligonucleotides, modificationof the internucleotide linkages or both modification of the sugar andthe internucleotide linkage.

A particularly useful group of nucleotides for use in increasingnuclease resistance at the termini of oligonucleotides are those having2′-O-alkylamino groups thereon. The amino groups of such nucleotides canbe groups that are protonated at physiological pH. These include amines,monoalkyl substituted amines, dialkyl substituted amines andheterocyclic amines such as imidazole. Particularly useful are the loweralkyl amines including 2′-O-ethylamine and 2′-O-propylamine. SuchO-alkylamines can also be included on the 3′ position of the 3′ terminusnucleotide. Thus the 3′ terminus nucleotide could include both a 2′ anda 3′-alkylamino substituent.

In selecting for nuclease resistance, it is important not to detractfrom binding affinity. Certain phosphorus based linkage have been shownto increase nuclease resistance. The above described phosphorothioatelinkage increase nuclease resistance, however, it also causes loss ofbinding affinity. Thus, generally for use in this invention, ifphosphorothioate internucleotide linkage are used, other modificationwill be made to nucleotide units that increase binding affinity tocompensate for the decreased affinity contribute by the phosphorothioatelinkages.

Other phosphorus based linkages having increase nuclease resistance thatdo not detract from binding affinity include 3′-methylene phosphonatesand 3′-deoxy-3′-amino-phosphoroamidate linkages. A further class oflinkages that contribute nuclease resistance but do not detract frombinding affinity are non-phosphate in nature. Preferred among these aremethylene(methylimino) linkages, dimethylhydraxino linkages, and amine 3and amide 4 linkages as described (Freier and Altmann, Nucleic AcidResearch, 1997, 25, 4429-4443).

J. There are a number of potential items to consider when designingoligonucleotides having improved binding affinities. It appears that oneeffective approach to constructing modified oligonucleotides with veryhigh RNA binding affinity is the combination of two or more differenttypes of modifications, each of which contributes favorably to variousfactors that might be important for binding affinity.

Freier and Altmann, Nucleic Acids Research, (1997) 25:4429-4443,recently published a study on the influence of structural modificationsof oligonucleotides on the stability of their duplexes with target RNA.In this study, the authors reviewed a series of oligonucleotidescontaining more than 200 different modifications that had beensynthesized and assessed for their hybridization affinity and T_(m).Sugar modifications studied included substitutions on the 2′-position ofthe sugar, 3′-substitution, replacement of the 4′-oxygen, the use ofbicyclic sugars, and four member ring replacements. Several nucleobasemodifications were also studied including substitutions at the 5, or 6position of thymine, modifications of pyrimidine heterocycle andmodifications of the purine heterocycle. Numerous backbone modificationswere also investigated including backbones bearing phosphorus, backbonesthat did not bear a phosphorus atom, and backbones that were neutral.

Four general approaches might be used to improve hybridization ofoligonucleotides to RNA targets. These include: preorganization of thesugars and phosphates of the oligodeoxynucleotide strand intoconformations favorable for hybrid formation, improving stacking ofnucleobases by the addition of polarizable groups to the heterocyclebases of the nucleotides of the oligonucleotide, increasing the numberof H-bonds available for A-U pairing, and neutralization of backbonecharge to facilitate removing undesirable repulsive interactions. Wehave found that by utilizing the first of these, preorganization of thesugars and phosphates of the oligodeoxynucleotide strand intoconformations favorable for hybrid formation, to be a preferred methodto achieve improve binding affinity. It can further be used incombination with the other three approaches.

Sugars in DNA:RNA hybrid duplexes frequently adopt a C3′ endoconformation. Thus modifications that shift the conformationalequilibrium of the sugar moieties in the single strand toward thisconformation should preorganize the antisense strand for binding to RNA.Of the several sugar modifications that have been reported and studiedin the literature, the incorporation of electronegative substituentssuch as 2′-fluoro or 2′-alkoxy shift the sugar conformation towards the3′ endo (northern) pucker conformation. This preorganizes anoligonucleotide that incorporates such modifications to have an A-formconformational geometry. This A-form conformation results in increasedbinding affinity of the oligonucleotide to a target RNA strand.

As used herein, the terms “substituent” and “substituent group” refersto groups that are attached to nucleosides of the invention. Substituentgroups are preferably attached to selected sugar moieties but canalternatively be attached to selected heterocyclic base moieties.Selected nucleosides may have substituent groups at both theheterocyclic base and the sugar moiety, however a single substituentgroup is preferred at a sugar 2′,3′ or 5′-positions with the 2′-positionbeing particularly preferred.

Substituent groups include fluoro, O-alkyl, O-alkylamino, O-alkylalkoxy,O-alkylaminoalkyl, O-alkyl imidazole, and polyethers of the formula(O-alkyl)_(m), where m is 1 to about 10. Preferred among thesepolyethers are linear and cyclic polyethylene glycols (PEGs), and(PEG)-containing groups, such as crown ethers and those which aredisclosed by Ouchi et al. (Drug Design and Discovery 1992, 9, 93),Ravasio et al. (J. Org. Chem. 1991, 56, 4329) and Delgardo et. al.(Critical Reviews in Therapeutic Drug Carrier Systems 1992, 9, 249),each of which is herein incorporated by reference in its entirety.Further sugar modifications are disclosed in Cook, P. D., Anti-CancerDrug Design, 1991, 6, 585-607. Fluoro, O-alkyl, O-alkylamino, O-alkylimidazole, O-alkylaminoalkyl, and alkyl amino substitution is describedin U.S. patent application Ser. No. 08/398,901, filed Mar. 6, 1995,entitled Oligomeric Compounds having Pyrimidine Nucleotide(s) with 2′and 5′ Substitutions, hereby incorporated by reference in its entirety.

Additional substituent groups amenable to the present invention include—SR and —NR₂ groups, wherein each R is, independently, hydrogen, aprotecting group or substituted or unsubstituted alkyl, alkenyl, oralkynyl. 2′-SR nucleosides are disclosed in U.S. Pat. No. 5,670,633,issued Sep. 23, 1997, hereby incorporated by reference in its entirety.The incorporation of 2′-SR monomer synthons are disclosed by Hamm etal., J. Org. Chem., 1997, 62, 3415-3420. 2′-NR₂ nucleosides aredisclosed by Goettingen, M., J. Org. Chem., 1996, 61, 6273-6281; andPolushin et al., Tetrahedron Lett., 1996, 3 7, 3227-3230.

Further representative substituent groups include hydrogen, hydroxyl,C₁-C₂₀ alkyl, C₂-C₂₀ alkenyl, C₂-C₂₀ alkynyl, halogen, amino, thiol,keto, carboxyl, nitro, nitroso, nitrile, trifluoromethyl,trifluoromethoxy, O-alkyl, O-alkenyl, O-alkynyl, S-alkyl, S-alkenyl,S-alkynyl, NH-alkyl, NH-alkenyl, NH-alkynyl, N-dialkyl, O-aryl, S-aryl,NH-aryl, O-aralkyl, S-aralkyl, NH-aralkyl, N-phthalimido, imidazole,azido, hydrazino, hydroxylamino, isocyanato, sulfoxide, sulfone,sulfide, disulfide, silyl, aryl, heterocycle, carbocycle, intercalator,reporter molecule, conjugate, polyamine, polyamide, polyalkylene glycol,or polyether;

or each substituent group has one of formula I or II:

wherein:

Z₀ is O, S or NH;

J is a single bond, O or C(═O);

E is C₁-C₁₀ alkyl, N(R₁)(R₂), N(R₁)(R₅), N═C(R₁)(R₂), N═C(R₁)(R₅) or hasone of formula III or IV;

each R₆, R₇, R₈, R₉ and R₁₀ is, independently, hydrogen, C(O)R₁₁,substituted or unsubstituted C₁-C₁₀ alkyl, substituted or unsubstitutedC₂-C₁₀ alkenyl, substituted or unsubstituted C₂-C₁₀ alkynyl,alkylsulfonyl, arylsulfonyl, a chemical functional group or a conjugategroup, wherein the substituent groups are selected from hydroxyl, amino,alkoxy, carboxy, benzyl, phenyl, nitro, thiol, thioalkoxy, halogen,alkyl, aryl, alkenyl and alkynyl;

or optionally, R₇ and R₈, together form a phthalimido moiety with thenitrogen atom to which they are attached;

or optionally, R₉ and R₁₀, together form a phthalimido moiety with thenitrogen atom to which they are attached;

each R₁₁ is, independently, substituted or unsubstituted C₁-C₁₀ alkyl,trifluoromethyl, cyanoethyloxy, methoxy, ethoxy, t-butoxy, allyloxy,9-fluorenylmethoxy, 2-(trimethylsilyl)-ethoxy, 2,2,2-trichloroethoxy,benzyloxy, butyryl, iso-butyryl, phenyl or aryl;

R₅ is T-L,

T is a bond or a linking moiety;

L is a chemical functional group, a conjugate group or a solid supportmaterial;

each R₁ and R₂ is, independently, H, a nitrogen protecting group,substituted or unsubstituted C₁-C₁₀ alkyl, substituted or unsubstitutedC₂-C₁₀ alkenyl, substituted or unsubstituted C₂-C₁₀ alkynyl, whereinsaid substitution is OR₃, SR₃, NH₃ ⁺, N(R₃)(R₄), guanidino or acyl wheresaid acyl is an acid amide or an ester;

or R₁ and R₂, together, are a nitrogen protecting group or are joined ina ring structure that optionally includes an additional heteroatomselected from N and O;

or R₁, T and L, together, are a chemical functional group;

each R₃ and R₄ is, independently, H, C₁-C₁₀ alkyl, a nitrogen protectinggroup, or R₃ and R₄, together, are a nitrogen protecting group;

or R₃ and R₄ are joined in a ring structure that optionally includes anadditional heteroatom selected from N and O;

Z₄ is OX, SX, or N(X)₂;

each X is, independently, H, C₁-C₈ alkyl, C₁-C₉ haloalkyl, C(═NH)N(H)R₅,C(═O)N(H)R₅ or OC(═O)N(H)R₅;

R₅ is H or C₁-C₈ alkyl;

Z₁, Z₂ and Z₃ comprise a ring system having from about 4 to about 7carbon atoms or having from about 3 to about 6 carbon atoms and 1 or 2hetero atoms wherein said hetero atoms are selected from oxygen,nitrogen and sulfur and wherein said ring system is aliphatic,unsaturated aliphatic, aromatic, or saturated or unsaturatedheterocyclic;

Z₅ is alkyl or haloalkyl having 1 to about 10 carbon atoms, alkenylhaving 2 to about 10 carbon atoms, alkynyl having 2 to about 10 carbonatoms, aryl having 6 to about 14 carbon atoms, N(R₁)(R₂) OR₁ halo, SR₁or CN;

each q₁ is, independently, an integer from 1 to 10;

each q₂ is, independently, 0 or 1;

q₃ is 0 or an integer from 1 to 10;

q₄ is an integer from 1 to 10;

q₅ is from 0, 1 or 2; and provided that when q₃ is 0, q₄ is greater than1.

Representative substituents groups of Formula I are disclosed in U.S.patent application Ser. No. 09/130,973, filed Aug. 7, 1998, entitled“Capped 2′-Oxyethoxy Oligonucleotides,” hereby incorporated by referencein its entirety.

Representative cyclic substituent groups of Formula II are disclosed inU.S. patent application Ser. No. 09/123,108, filed Jul. 27, 1998,entitled “RNA Targeted 2′-Modified Oligonucleotides that areConformationally Preorganized,” hereby incorporated by reference in itsentirety.

Particularly preferred substituent groups include O[(CH₂)_(n)O]_(m)CH₃,O(CH₂)_(n)OCH₃, O(CH₂)_(n)NH₂, O(CH₂)_(n)CH₃, O(CH₂)_(n)ONH₂, andO(CH₂)_(n)ON[(CH₂)_(n)CH₃)]₂, where n and m are from 1 to about 10.

Some preferred oligomeric compounds of the invention contain, at leastone nucleoside having one of the following substituent groups: C₁ to C₁₀lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl orO-aralkyl, SH, SCH₃, OCN, Cl, Br, CN, CF₃, OCF₃, SOCH₃, SO₂CH₃, ONO₂,NO₂, N₃, NH₂, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino,polyalkylamino, substituted silyl, an RNA cleaving group, a reportergroup, an intercalator, a group for improving the pharmacokineticproperties of an oligomeric compound, or a group for improving thepharmacodynamic properties of an oligomeric compound, and othersubstituents having similar properties. A preferred modificationincludes 2′-methoxyethoxy [2′-O—CH₂CH₂OCH₃, also known as2′-O-(2-methoxyethyl) or 2′-MOE] (Martin et al., Helv. Chim. Acta, 1995,78, 486), i.e., an alkoxyalkoxy group. A further preferred modificationis 2′-dimethylaminooxyethoxy, i.e., a O(CH₂)_(2ON(CH) ₃)₂ group, alsoknown as 2′-DMAOE. Representative aminooxy substituent groups aredescribed in co-owned U.S. patent application Ser. No. 09/344,260, filedJun. 25, 1999, entitled “Aminooxy-Functionalized Oligomers”; and a U.S.patent application entitled “Aminooxy-Functionalized Oligomers andMethods for Making Same,” filed Aug. 9, 1999, presently identified byattorney docket number ISIS-3993, hereby incorporated by reference intheir entirety.

Other preferred modifications include 2′-methoxy (2′-O—CH₃),2′-aminopropoxy (2′-OCH₂CH₂CH₂NH₂) and 2′-fluoro (2′-F). Similarmodifications may also be made at other positions on nucleosides andoligomers, particularly the 3′ position of the sugar on the 3′ terminalnucleoside or in 2′-5′ linked oligomers and the 5′ position of 5′terminal nucleoside. Oligomers may also have sugar mimetics such ascyclobutyl moieties in place of the pentofuranosyl sugar. RepresentativeUnited States patents that teach the preparation of such modified sugarsstructures include, but are not limited to, U.S. Pat. Nos. 4,981,957;5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786;5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909;5,610,300; 5,627,0531 5,639,873; 5,646,265; 5,658,873; 5,670,633; and5,700,920, certain of which are commonly owned, and each of which isherein incorporated by reference, and commonly owned U.S. patentapplication Ser. No. 08/468,037, filed on Jun. 5, 1995, also hereinincorporated by reference.

Representative guanidino substituent groups that are shown in formulaIII and IV are disclosed in co-owned U.S. patent application Ser. No.09/349,040 entitled “Functionalized Oligomers,” filed Jul. 7, 1999,hereby incorporated by reference in its entirety.

Representative acetamido substituent groups are disclosed in a U.S.patent application entitled “2′-O-Acetamido Modified Monomers andOligomers,” filed Aug. 19, 1999, presently identified by attorney docketnumber ISIS-4071, hereby incorporated by reference in its entirety.

Representative dimethylaminoethyloxyethyl substituent groups aredisclosed in an International Patent Application entitled“2′-O-Dimethylaminoethyloxyethyl-Modified Oligonucleotides,” filed Aug.6, 1999, presently identified by attorney docket number ISIS-4045,hereby incorporated by reference in its entirety.

Several 2′-substituents confer a 3′-endo pucker to the sugar where theyare incorporated. This pucker conformation further assists in increasingthe Tm of the oligonucleotide with its target.

The high binding affinity resulting from 2′ substitution has beenpartially attributed to the 2′ substitution causing a C3′ endo sugarpucker which in turn may give the oligomer a favorable A-form likegeometry. This is a reasonable hypothesis since substitution at the 2′position by a variety of electronegative groups (such as fluoro andO-alkyl chains) has been demonstrated to cause C3′ endo sugar puckering(De Mesmaeker et al., Acc. Chem. Res., 1995, 28, 366-374; Lesnik et al.,Biochemistry, 1993, 32, 7832-7838).

In addition, for 2′-substituents containing an ethylene glycol motif, agauche interaction between the oxygen atoms around the O—C—C—O torsionof the side chain may have a stabilizing effect on the duplex (Freier etal.,Nucleic Acids Research, (1997) 25:4429-4442). Such gaucheinteractions have been observed experimentally for a number of years(Wolfe et al., Acc. Chem. Res., 1972, 5, 102; Abe et al., J. Am. Chem.Soc., 1976, 98, 468). This gauche effect may result in a configurationof the side chain that is favorable for duplex formation. The exactnature of this stabilizing configuration has not yet been explained.While we do not want to be bound by theory, it may be that holding theO—C—C—O torsion in a single gauche configuration, rather than a morerandom distribution seen in an alkyl side chain, provides an entropicadvantage for duplex formation.

To better understand the higher RNA affinity of 2′-O-methoxyethylsubstituted RNA and to examine the conformational properties of the2′-O-methoxyethyl substituent, a self-complementary dodecameroligonucleotide

2′-O-MOE r(CGCGAAUUCGCG) SEQ ID NO: 1

was synthesized, crystallized and its structure at a resolution of 1.7Angstrom was determined. The crystallization conditions used were 2 mMoligonucleotide, 50 mM Na Hepes pH 6.2-7.5, 10.50 mM MgCl₂, 15% PEG 400.The crystal data showed: space group C2, cell constants a=41.2 Å, b=34.4Å, c=46.6 Å, =92.4°. The resolution was 1.7 Å at −170° C. The currentR=factor was 20% (R_(free) 26%).

This crystal structure is believed to be the first crystal structure ofa fully modified RNA oligonucleotide analogue. The duplex adopts anoverall A-form conformation and all modified sugars display C3′-endopucker. In most of the 2′-O-substituents, the torsion angle around theA′-B′ bond, as depicted in Structure II below, of the ethylene glycollinker has a gauche conformation. For 2′-O-MOE, A′ and B′ of StructureII below are methylene moieties of the ethyl portion of the MOE and R′is the methoxy portion.

In the crystal, the 2′-O-MOE RNA duplex adopts a general orientationsuch that the crystallographic 2-fold rotation axis does not coincidewith the molecular 2-fold rotation axis. The duplex adopts the expectedA-type geometry and all of the 24 2′-O-MOE substituents were visible inthe electron density maps at full resolution. The electron density mapsas well as the temperature factors of substituent atoms indicateflexibility of the 2′-O-MOE substituent in some cases.

Most of the 2′-O-MOE substituents display a gauche conformation aroundthe C—C bond of the ethyl linker. However, in two cases, a transconformation around the C—C bond is observed. The lattice interactionsin the crystal include packing of duplexes against each other via theirminor grooves. Therefore, for some residues, the conformation of the2′-O-substituent is affected by contacts to an adjacent duplex. Ingeneral, variations in the conformation of the substituents (e.g. g⁺ org⁻ around the C—C bonds) create a range of interactions betweensubstituents, both inter-strand, across the minor groove, andintra-strand. At one location, atoms of substituents from two residuesare in van der Waals contact across the minor groove. Similarly, a closecontact occurs between atoms of substituents from two adjacentintra-strand residues.

Previously determined crystal structures of A-DNA duplexes were forthose that incorporated isolated 2′-O-methyl T residues. In the crystalstructure noted above for the 2′-O-MOE substituents, a conservedhydration pattern has been observed for the 2′-O-MOE residues. A singlewater molecule is seen located between O2′, O3′ and the methoxy oxygenatom of the substituent, forming contacts to all three of between 2.9and 3.4 A. In addition, oxygen atoms of substituents are involved inseveral other hydrogen bonding contacts. For example, the methoxy oxygenatom of a particular 2′-O-substituent forms a hydrogen bond to N3 of anadenosine from the opposite strand via a bridging water molecule.

In several cases a water molecule is trapped between the oxygen atomsO2′, O3′ and OC′ of modified nucleosides. 2′-O-MOE substituents withtrans conformation around the C—C bond of the ethylene glycol linker areassociated with close contacts between OC′ and N2 of a guanosine fromthe opposite strand, and, water-mediated, between OC′ and N3(G). Whencombined with the available thermodynamic data for duplexes containing2′-O-MOE modified strands, this crystal structure allows for furtherdetailed structure-stability analysis of other antisense modifications.

In extending the crystallographic structure studies, molecular modelingexperiments were performed to study further enhanced binding affinity ofoligonucleotides having 2′-O-modifications of the invention. Thecomputer simulations were conducted on compounds of SEQ ID NO: 1, above,having 2′-O-modifications of the invention located at each of thenucleoside of the oligonucleotide. The simulations were performed withthe oligonucleotide in aqueous solution using the AMBER force fieldmethod (Cornell et al., J. Am. Chem. Soc., 1995, 117,5179-5197)(modeling software package from UCSF, San Francisco, Calif.).The calculations were performed on an Indigo2 SGI machine (SiliconGraphics, Mountain View, Calif.).

Further 2′-O-modifications of the inventions include those having a ringstructure that incorporates a two atom portion corresponding to the A′and B′ atoms of Structure II. The ring structure is attached at the 2′position of a sugar moiety of one or more nucleosides that areincorporated into an oligonucleotide. The 2′-oxygen of the nucleosidelinks to a carbon atom corresponding to the A′ atom of Structure II.These ring structures can be aliphatic, unsaturated aliphatic, aromaticor heterocyclic. A further atom of the ring (corresponding to the B′atom of Structure II), bears a further oxygen atom, or a sulfur ornitrogen atom. This oxygen, sulfur or nitrogen atom is bonded to one ormore hydrogen atoms, alkyl moieties, or haloalkyl moieties, or is partof a further chemical moiety such as a ureido, carbamate, amide oramidine moiety. The remainder of the ring structure restricts rotationabout the bond joining these two ring atoms. This assists in positioningthe “further oxygen, sulfur or nitrogen atom” (part of the R position asdescribed above) such that the further atom can be located in closeproximity to the 3′-oxygen atom (O3′) of the nucleoside.

The ring structure can be further modified with a group useful formodifying the hydrophilic and hydrophobic properties of the ring towhich it is attached and thus the properties of an oligonucleotide thatincludes the 2′-O-modifications of the invention. Further groups can beselected as groups capable of assuming a charged structure, e.g. anamine. This is particularly useful in modifying the overall charge of anoligonucleotide that includes a 2′-O-modifications of the invention.When an oligonucleotide is linked by charged phosphate groups, e.g.phosphorothioate or phosphodiester linkages, location of a counter ionon the 2′-O-modification, e.g. an amine functionality, locallynaturalizes the charge in the local environment of the nucleotidebearing the 2′-O-modification. Such neutralization of charge willmodulate uptake, cell localization and other pharmacokinetic andpharmacodynamic effects of the oligonucleotide.

Preferred ring structures of the invention for inclusion as a 2′-Omodification include cyclohexyl, cyclopentyl and phenyl rings as well asheterocyclic rings having spacial footprints similar to cyclohexyl,cyclopentyl and phenyl rings. Particularly preferred 2′-O-substituentgroups of the invention are listed below including an abbreviation foreach:

2′-O-(trans 2-methoxy cyclohexyl)—2′-O-(TMCHL)

2′-O-(trans 2-methoxy cyclopentyl)—2′-O-(TMCPL)

2′-O-(trans 2-ureido cyclohexyl)—2′-O-(TUCHL)

2′-O-(trans 2-methoxyphenyl)—2′-O-(2MP)

Structural details for duplexes incorporating such 2-O-substituents wereanalyzed using the described AMBER force field program on the Indigo2SGI machine. The simulated structure maintained a stable A-form geometrythroughout the duration of the simulation. The presence of the 2′substitutions locked the sugars in the C3′-endo conformation.

The simulation for the TMCHL modification revealed that the 2′-O-(TMCHL)side chains have a direct interaction with water molecules solvating theduplex. The oxygen atoms in the 2′-O-(TMCHL) side chain are capable offorming a water-mediated interaction with the 3′ oxygen of the phosphatebackbone. The presence of the two oxygen atoms in the 2′-O-(TMCHL) sidechain gives rise to favorable gauche interactions. The barrier forrotation around the O—C—C—O torsion is made even larger by this novelmodification. The preferential preorganization in an A-type geometryincreases the binding affinity of the 2′-O-(TMCHL) to the target RNA.The locked side chain conformation in the 2′-O-(TMCHL) group created amore favorable pocket for binding water molecules. The presence of thesewater molecules played a key role in holding the side chains in thepreferable gauche conformation. While not wishing to be bound by theory,the bulk of the substituent, the diequatorial orientation of thesubstituents in the cyclohexane ring, the water of hydration and thepotential for trapping of metal ions in the conformation generated willadditionally contribute to improved binding affinity and nucleaseresistance of oligonucleotides incorporating nucleosides having this2′-O-modification.

As described for the TMCHL modification above, identical computersimulations of the 2′-O-(TMCPL), the 2′-O-(2MP) and 2′-O-(TUCHL)modified oligonucleotides in aqueous solution also illustrate thatstable A-form geometry will be maintained throughout the duration of thesimulation. The presence of the 2′ substitution will lock the sugars inthe C3′-endo conformation and the side chains will have directinteraction with water molecules solvating the duplex. The oxygen atomsin the respective side chains are capable of forming a water-mediatedinteraction with the 3′ oxygen of the phosphate backbone. The presenceof the two oxygen atoms in the respective side chains give rise to thefavorable gauche interactions. The barrier for rotation around therespective O—C—C—O torsions will be made even larger by respectivemodification. The preferential preorganization in A-type geometry willincrease the binding affinity of the respective 2′-O-modifiedoligonucleotides to the target RNA. The locked side chain conformationin the respective modifications will create a more favorable pocket forbinding water molecules. The presence of these water molecules plays akey role in holding the side chains in the preferable gaucheconformation. The bulk of the substituent, the diequatorial orientationof the substituents in their respective rings, the water of hydrationand the potential trapping of metal ions in the conformation generatedwill all contribute to improved binding affinity and nuclease resistanceof oligonucleotides incorporating nucleosides having these respective2′-O-modification.

Preferred for use as the B-form nucleotides for eliciting RNase H areribonucleotides having 2′-deoxy-2′-S-methyl, 2′-deoxy-2′-methyl,2′-deoxy-2′-amino, 2′-deoxy-2′-mono or dialkyl substituted amino,2′-deoxy-2′-fluoromethyl, 2′-deoxy-2′-difluoromethyl,2′-deoxy-2′-trifluoromethyl, 2′-deoxy-2′-methylene,2′-deoxy-2′-fluoromethylene, 2′-deoxy-2′-difluoromethylene,2′-deoxy-2′-ethyl, 2′-deoxy-2′-ethylene and 2′-deoxy-2′-acetylene. Thesenucleotides can alternately be described as 2′-SCH₃ ribonucleotide,2′-CH₃ ribonucleotide, 2′-NH₂ ribonucleotide 2′-NH(C₁-C₂ alkyl)ribonucleotide, 2′-N(C₁-C₂ alkyl)₂ ribonucleotide, 2′-CH₂Fribonucleotide, 2′-CHF₂ ribonucleotide, 2′-CF₃ ribonucleotide, 2′=CH₂ribonucleotide, 2′=CHF ribonucleotide, 2′=CF₂ ribonucleotide, 2′-C₂H₅ribonucleotide, 2′-CH═CH₂ ribonucleotide, 2′-CCH ribonucleotide. Afurther useful ribonucleotide is one having a ring located on the ribosering in a cage-like structure including3′,O,4′-C-methyleneribonucleotides. Such cage-like structures willphysically fix the ribose ring in the desired conformation.

Additionally, preferred for use as the B-form nucleotides for elicitingRNase H are arabino nucleotides having 2′-deoxy-2′-cyano,2′-deoxy-2′-fluoro, 2′-deoxy-2′-chloro, 2′-deoxy-2′-bromo,2′-deoxy-2′-azido, 2′-methoxy and the unmodified arabino nucleotide(that includes a 2′-OH projecting upwards towards the base of thenucleotide). These arabino nucleotides can alternately be described as2′-CN arabino nucleotide, 2′-F arabino nucleotide, 2′-Cl arabinonucleotide, 2′-Br arabino nucleotide, 2′-N₃ arabino nucleotide, 2′-O—CH₃arabino nucleotide and arabino nucleotide.

Such nucleotides are linked together via phosphorothioate,phosphorodithioate, boranophosphate or phosphodiester linkages,particularly preferred is the phosphorothioate linkage.

Illustrative of the B-form nucleotides for use in the invention is a2′-S-methyl (2′-SMe) nucleotide that resides in C2′ endo conformation.It can be compared to 2′-O-methyl (2′-OMe)nucleotides that resides in aC3′ endo conformation. Particularly suitable for use in comparing thesetwo nucleotides are molecular dynamic investigations using a SGI[Silicon Graphics, Mountain View, Calif.] computer and the AMBER [UCSF,San Francisco, Calif.] modeling software package for computersimulations.

Ribose conformations in C2′-modified nucleosides containing S-methylgroups were examined. To understand the influence of 2′-O-methyl and2′-S-methyl groups on the conformation of nucleosides, we evaluated therelative energies of the 2′-O- and 2′-S-methylguanosine, along withnormal deoxyguanosine and riboguanosine, starting from both C2′-endo andC3′-endo conformations using ab initio quantum mechanical calculations.All the structures were fully optimized at HF/6-31G* level and singlepoint energies with electron-correlation were obtained at theMP2/6-31G*//HF/6-31G* level. As shown in Table 1, the C2′-endoconformation of deoxyguanosine is estimated to be 0.6 kcal/mol morestable than the C3′-endo conformation in the gas-phase. Theconformational preference of the C2′-endo over the C3′-endo conformationappears to be less dependent upon electron correlation as revealed bythe MP2/6-31G*//HF/6-31G* values which also predict the same differencein energy. The opposite trend is noted for riboguanosine. At theHF/6-31G* and MP2/6-31G*//HF/6-31G* levels, the C3′-endo form ofriboguanosine is shown to be about 0.65 and 1.41 kcal/mol more stablethan the C2′endo form, respectively. TABLE 1 Relative energies* of theC3′-endo and C2′-endo conformations of representative nucleosides.CONTINUUM HF/6-31G MP2/6-31-G MODEL AMBER dG 0.60 0.56 0.88 0.65 rG−0.65 −1.41 −0.28 −2.09 2′-O-MeG −0.89 −1.79 −0.36 −0.86 2′-S-MeG 2.551.41 3.16 2.43*energies are in kcal/mol relative to the C2′-endo conformation

Table 1 also includes the relative energies of 2′-O-methylguanosine and2′-S-methylguanosine in C2′-endo and C3′-endo conformation. This dataindicates the electronic nature of C2′-substitution has a significantimpact on the relative stability of these conformations. Substitution ofthe 2′-O-methyl group increases the preference for the C3′-endoconformation (when compared to riboguanosine) by about 0.4 kcal/mol atboth the HF/6-31G* and MP2/6-31G*//HF/6-31G* levels. In contrast, the2′-S-methyl group reverses the trend. The C2′-endo conformation isfavored by about 2.6 kcal/mol at the HF/6-31G* level, while the samedifference is reduced to 1.41 kcal/mol at the MP2/6-31G*//HF/6-31G*level. For comparison, and also to evaluate the accuracy of themolecular mechanical force-field parameters used for the 2′-O-methyl and2′-S-methyl substituted nucleosides, we have calculated the gas phaseenergies of the nucleosides. The results reported in Table 1 indicatethat the calculated relative energies of these nucleosides comparequalitatively well with the ab initio calculations.

Additional calculations were also performed to gauge the effect ofsolvation on the relative stability of nucleoside conformations. Theestimated solvation effect using HF/6-31G* geometries confirms that therelative energetic preference of the four nucleosides in the gas-phaseis maintained in the aqueous phase as well (Table 1). Solvation effectswere also examined using molecular dynamics simulations of thenucleosides in explicit water. From these trajectories, one can observethe predominance of C2′-endo conformation for deoxyriboguanosine and2′-S-methylriboguanosine while riboguanosine and2′-O-methylriboguanosine prefer the C3′-endo conformation. These resultsare in much accord with the available NMR results on2′-S-methylribonucleosides. NMR studies of sugar puckering equilibriumusing vicinal spin-coupling constants have indicated that theconformation of the sugar ring in 2′-S-methylpyrimidine nucleosides showan average of >75% S-character, whereas the corresponding purine analogsexhibit an average of >90% S-pucker [Fraser, A., Wheeler, P., Cook, P.D. and Sanghvi, Y. S., J. Heterocycl. Chem., 1993, 30, 1277-1287]. Itwas observed that the 2′-S-methyl substitution in deoxynucleosideconfers more conformational rigidity to the sugar conformation whencompared with deoxyribonucleosides.

Structural features of DNA:RNA, OMe_DNA:RNA and SMe_DNA:RNA hybrids werealso observed. The average RMS deviation of the DNA:RNA structure fromthe starting hybrid coordinates indicate the structure is stabilizedover the length of the simulation with an approximate average RMSdeviation of 1.0 Å. This deviation is due, in part, to inherentdifferences in averaged structures (i.e. the starting conformation) andstructures at thermal equilibrium. The changes in sugar puckerconformation for three of the central base pairs of this hybrid are ingood agreement with the observations made in previous NMR studies. Thesugars in the RNA strand maintain very stable geometries in the C3′-endoconformation with ring pucker values near 0°. In contrast, the sugars ofthe DNA strand show significant variability.

The average RMS deviation of the OMe_DNA:RNA is approximately 1.2 Å fromthe starting A-form conformation; while the SMe_DNA:RNA shows a slightlyhigher deviation (approximately 1.8 Å) from the starting hybridconformation. The SMe_DNA strand also shows a greater variance in RMSdeviation, suggesting the S-methyl group may induce some structuralfluctuations. The sugar puckers of the RNA complements maintain C3′-endo puckering throughout the simulation. As expected from thenucleoside calculations, however, significant differences are noted inthe puckering of the OMe_DNA and SMe_DNA strands, with the formeradopting C3′-endo, and the latter, C1′-exo/C2′-endo conformations.

An analysis of the helicoidal parameters for all three hybrid structureshas also been performed to further characterize the duplex conformation.Three of the more important axis-basepair parameters that distinguishthe different forms of the duplexes, X-displacement, propeller twist,and inclination, are reported in Table 2. Usually, an X-displacementnear zero represents a B-form duplex; while a negative displacement,which is a direct measure of deviation of the helix from the helicalaxis, makes the structure appear more A-like in conformation. In A-formduplexes, these values typically vary from −4 Å to −5Å. In comparingthese values for all three hybrids, the SMe_DNA:RNA hybrid shows themost deviation from the A-form value, the OMe_DNA:RNA shows the least,and the DNA:RNA is intermediate. A similar trend is also evident whencomparing the inclination and propeller twist values with ideal A-formparameters. These results are further supported by an analysis of thebackbone and glycosidic torsion angles of the hybrid structures.Glycosidic angles (X) of A-form geometries, for example, are typicallynear −159E while B form values are near −102E. These angles are found tobe −162E, −133E, and −108E for the OMe_DNA, DNA, and SMe_DNA strands,respectively. All RNA complements adopt an X angle close to −160E. Inaddition, “crankshaft” transitions were also noted in the backbonetorsions of the central UpU steps of the RNA strand in the SMe_DNA:RNAand DNA;RNA hybrids. Such transitions suggest some local conformationalchanges may occur to relieve a less favorable global conformation. Takenoverall, the results indicate the amount of A-character decreases asOMe_DNA:RNA>DNA:RNA>SMe_DNA:RNA, with the latter two adopting moreintermediate conformations when compared to A- and B-form geometries.TABLE 2 Average helical parameters derived from the last 500 ps ofsimulation time. (canonical A- and B-form values are given forcomparison) Helicoidal B-DNA B-DNA A-DNA Parameter (x-ray) (fibre)(fibre) DNA:RNA OMe_DNA:RNA SMe_DNA:RNA X-disp 1.2 0.0 −5.3 −4.5 −5.4−3.5 Inclination −2.3 1.5 20.7 11.6 15.1 0.7 Propeller −16.4 −13.3 −7.5−12.7 −15.8 −10.3

Stability of C2′-modified DNA:RNA hybrids was determined. Although theoverall stability of the DNA:RNA hybrids depends on several factorsincluding sequence-dependencies and the purine content in the DNA or RNAstrands DNA:RNA hybrids are usually less stable than RNA:RNA duplexesand, in some cases, even less stable than DNA:DNA duplexes. Availableexperimental data attributes the relatively lowered stability of DNA:RNAhybrids largely to its intermediate conformational nature betweenDNA:DNA (B-family) and RNA:RNA (A-family) duplexes. The overallthermodynamic stability of nucleic acid duplexes may originate fromseveral factors including the conformation of backbone, base-pairing andstacking interactions. While it is difficult to ascertain the individualthermodynamic contributions to the overall stabilization of the duplex,it is reasonable to argue that the major factors that promote increasedstability of hybrid duplexes are better stacking interactions(electrostatic B—B_interactions) and more favorable groove dimensionsfor hydration. The C2′-S-methyl substitution has been shown todestabilize the hybrid duplex. The notable differences in the risevalues among the three hybrids may offer some explanation. While the2′-S-methyl group has a strong influence on decreasing the base-stackingthrough high rise values (˜3.2 Å), the 2′-O-methyl group makes theoverall structure more compact with a rise value that is equal to thatof A-form duplexes (˜2.6 Å). Despite its overall A-like structuralfeatures, the SMe_DNA:RNA hybrid structure possesses an average risevalue of 3.2 Å which is quite close to that of B-family duplexes. Infact, some local base-steps (CG steps) may be observed to have unusuallyhigh rise values (as high as 4.5 Å). Thus, the greater destabilizationof 2′-S-methyl substituted DNA:RNA hybrids may be partly attributed topoor stacking interactions.

It has been postulated that RNase H binds to the minor groove of RNA:DNAhybrid complexes, requiring an intermediate minor groove width betweenideal A- and B-form geometries to optimize interactions between thesugar phosphate backbone atoms and RNase H. A close inspection of theaveraged structures for the hybrid duplexes using computer simulationsreveals significant variation in the minor groove width dimensions asshown in Table 3. Whereas the O-methyl substitution leads to a slightexpansion of the minor groove width when compared to the standardDNA:RNA complex, the S-methyl substitution leads to a generalcontraction (approximately 0.9 Å). These changes are most likely due tothe preferred sugar puckering noted for the antisense strands whichinduce either A- or B-like single strand conformations. In addition tominor groove variations, the results also point to potential differencesin the steric makeup of the minor groove. The O-methyl group points intothe minor groove while the S-methyl is directed away towards the majorgroove. Essentially, the S-methyl group has flipped through the basesinto the major groove as a consequence of C2′-endo puckering. TABLE 3Minor groove widths averaged over the last 500 ps of simulation timePhosphate DNA:RNA RNA:RNA Distance DNA:RNA OMe_DNA:RNA SMe_DNA:RNA(B-form) (A-form) P5-P20 15.27 16.82 13.73 14.19 17.32 P6-P19 15.5216.79 15.73 12.66 17.12 P7-P18 15.19 16.40 14.08 11.10 16.60 P8-P1715.07 16.12 14.00 10.98 16.14 P9-P16 15.29 16.25 14.98 11.65 16.93P10-P15 15.37 16.57 13.92 14.05 17.69

In addition to the modifications described above, the nucleotides of theoligonucleotides of the invention can have a variety of othermodification so long as these other modifications do not significantlydetract from the properties described above. Thus, for nucleotides thatare incorporated into oligonucleotides of the invention, thesenucleotides can have sugar portions that correspond tonaturally-occurring sugars or modified sugars. Representative modifiedsugars include carbocyclic or acyclic sugars, sugars having substituentgroups at their 2′ position, sugars having substituent groups at their3′ position, and sugars having substituents in place of one or morehydrogen atoms of the sugar. Other altered base moieties and alteredsugar moieties are disclosed in U.S. Pat. No. 3,687,808 and PCTapplication PCT/US89/02323.

Altered base moieties or altered sugar moieties also include othermodifications consistent with the spirit of this invention. Sucholigonucleotides are best described as being structurallydistinguishable from, yet functionally interchangeable with, naturallyoccurring or synthetic wild type oligonucleotides. All sucholigonucleotides are comprehended by this invention so long as theyfunction effectively to mimic the structure of a desired RNA or DNAstrand. A class of representative base modifications include tricycliccytosine analog, termed “G clamp” (Lin, et al., J. Am. Chem. Soc. 1998,120, 8531). This analog makes four hydrogen bonds to a complementaryguanine (G) within a helix by simultaneously recognizing theWatson-Crick and Hoogsteen faces of the targeted G. This G clampmodification when incorporated into phosphorothioate oligonucleotides,dramatically enhances antisense potencies in cell culture. Theoligonucleotides of the invention also can includephenoxazine-substituted bases of the type disclosed by Flanagan, et al.,Nat. Biotechnol. 1999, 17(1), 48-52.

Additional modifications may also be made at other positions on theoligonucleotide, particularly the 3′ position of the sugar on the 3′terminal nucleotide and the 5′ position of 5′ terminal nucleotide. Forexample, one additional modification of the oligonucleotides of theinvention involves chemically linking to the oligonucleotide one or moremoieties or conjugates which enhance the activity, cellular distributionor cellular uptake of the oligonucleotide. Such moieties include but arenot limited to lipid moieties such as a cholesterol moiety (Letsinger etal., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553), cholic acid (Manoharanet al., Bioorg. Med. Chem. Lett., 1994, 4, 1053), a thioether, e.g.,hexyl-S-tritylthiol (Manoharan et al., Ann. N. Y Acad. Sci., 1992, 660,306; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3, 2765), athiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20, 533), analiphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaraset al., EMBO J., 1991, 10, 111; Kabanov et al., FEBS Lett., 1990, 259,327; Svinarchuk et al., Biochimie, 1993, 75, 49), a phospholipid, e.g.,di-hexadecyl-rac-glycerol or triethylammonium1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al.,Tetrahedron Lett., 1995, 36, 3651; Shea et al., Nucl. Acids Res., 1990,18, 3777), a polyamine or a polyethylene glycol chain (Manoharan et al.,Nucleosides & Nucleotides, 1995, 14, 969), or adamantane acetic acid(Manoharan et al., Tetrahedron Lett., 1995, 36, 3651), a palmityl moiety(Mishra et al., Biochim. Biophys. Acta, 1995, 1264, 229), or anoctadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke etal., J. Pharmacol. Exp. Ther., 1996, 277, 923).

Human RNase H1 displays a strong positional preference for cleavage,i.e., it cleaves between 8-12 nucleotides from the 5N-RNA:3N-DNAterminus of the duplex. Within the preferred cleavage site, the enzymedisplays modest sequence preference with GU being a preferreddinucleotide. The minimum RNA:DNA duplex length that supports cleavageis 6-base pairs and the minimum RNA:DNA “gap size” that supportscleavage is 5-base pairs.

Properties of Purified Human RNase H1

The effects of various reaction conditions on the activity of HumanRNase H1 were evaluated (FIG. 1). The optimal pH for the enzyme in bothTris HCl and phosphate buffers was 7.0-8.0. At pH's above pH8.0, enzymeactivity was reduced. However, this could be due to instability of thesubstrate or effects on the enzyme, or both. To evaluate the potentialcontribution of changes in ionic strength to the activities observed atdifferent pHs, two buffers, NaHPO₄ and Tris HCl were studied at pH 7.0and gave the same enzyme activity even though the ionic strengthsdiffered. Enzyme activity was inhibited by increasing ionic strength(FIG. 1B) and N-ethymaleamide (FIG. 1C). Enzyme activity increased asthe temperature was raised from 25-42° C (FIG. 1D). Mg²⁺ stimulatedenzyme activity with an optimal concentration of 1 mM. At higherconcentrations, Mg²⁺ was inhibitory (FIG. 1E). In the presence of 1 mMMg²⁺, Mn²⁺ was inhibitory at all concentrations tested (FIG. 1F). Thepurified enzyme was quite stable and easily handled. In fact, the enzymecould be boiled and rapidly or slowly cooled without significant loss ofactivity (FIG. 1D). The initial rates of cleavage were determined forfour duplex substrates studied simultaneously. The initial rate ofcleavage for a phosphodiester DNA:RNA duplex was 1050±203 pmol L⁻min⁻(Table 4A). The initial rate of cleavage of a phosphorothioateoligodeoxynucleotide duplex was approximately four-fold faster than thatof the same duplex comprised of a phosphodiester antisenseoligodeoxynucleotide (Table 4A). The initial rates for 17-mer and 20-mersubstrates of different sequences were equal (Table 4B). However, when a25-mer heteroduplex, containing the 17-mer sequence in the center of theduplex was digested (RNA 3), the rate was 50% faster. Interestingly, theKm of the enzyme for the 25 mer duplex was 40% lower than that for the17 mer while the Vmax's for both duplexes were the same (see Table 6),suggesting that with the increase in length, a larger number of cleavagesites are available resulting in an increase in the number of productivebinding interactions between the enzyme and substrate. As a result, alower substrate concentration is required for the longer duplex toachieve a cleavage rate equal to that of the shorter duplex.

To better characterize the substrate specificity of Human RNase H1,duplexes in which the antisense oligonucleotide was modified in the2′-position were studied. As previously reported for E. coli RNase H1,Human RNase H1 was unable to cleave substrates with 2′-modifications atthe cleavage site of the antisense DNA strand or the sense RNA strand(Table 5). For example, the initial rate of cleavage of a duplexcontaining a phosphorothioate oligodeoxynucleotide and its complementwas 3400 pmol L⁻¹min⁻¹ while that of its 2′-propoxy modified analog wasundetectable (Table 5). A duplex comprised of a fully modified2′-methoxy antisense strand also failed to support any cleavage (Table5). The placement of 2′-methoxy modifications around a central region ofoligodeoxynucleotides reduced the initial rate (Table 5). The smallerthe central oligodeoxynucleotide “gap” the lower the initial rate. Thesmallest “gapmer” for which cleavage could be measured was a 5deoxynucleotide gap. These data are highly consistent with observationswe have previously reported for E. coli RNase H1 except that for thebacterial enzyme the minimum gap size was 4 deoxynucleotides.

The Km and Vmax of Human RNase H1 for three substrates are shown inTable 6. The Km valves for all three substrates were substantially lowerthan those of E. coli RNase H1 (Table 6). As previously reported for E.coli RNase H1, the Km for a phosphorothioate containing duplex was lowerthan that of a phosphodiester duplex. The Vmax of the human enzyme was30 fold lower than that of the E. coli enzyme. The Vmax for thephosphorothioate containing substrate was less than the phosphodiesterduplex. This is probably due to inhibition of the enzyme at higherconcentrations by excess phosphorothioate single strand oligonucleotideas the initial rate of cleavage for a phosphorothioate containing duplexwas, in fact, greater than the phosphodiester (Table 4)

Binding Affinity and Specificity

To evaluate the binding affinity of Human RNase H1, a competitivecleavage assay in which increasing concentrations of noncleavablesubstrates were added was used. Using this approach, the Ki is formallyequivalent to the Kd for the competing substrates. Of the noncleavablesubstrates studied, Lineweaver-Burk analyses demonstrated that allinhibitors shown in Table 7 were competitive (data not shown). A duplexcontaining a phosphodiester oligodeoxynucleotide hybridized to aphosphodiester 2′-methoxy oligonucleotide as the noncleavable substrateis considered most like DNA:RNA. Table 7 shows the results of thesestudies and compares them to previously reported results for the E. colienzyme performed under similar conditions. Clearly, the affinity of thehuman enzyme for its DNA:RNA like substrate (DNA:2′-O-Me) wassubstantially greater than that of the E. coli enzyme, consistent withthe differences observed in Km (Table 6).

E. coli RNase H1 displays approximately equal affinity for RNA:RNA,RNA:2′-O-Me and DNA:2′-O-Me duplexes (Table 7). The human enzymedisplays similar binding properties, but is more able to discriminatebetween various duplexes. For example, the Kd for RNA:RNA wasapproximately 5 fold lower than the Kd for DNA:2′-O-Me. This is furtherdemonstrated by the Kd for the RNA:2′F duplex. The Kd for the DNA:2′-Fduplex was slightly greater than for the RNA:2′-F duplex and the RNA:RNAduplex, but clearly lower than for other duplexes. Thus, both enzymescan be considered double strand RNA binding proteins. However, HumanRNase H1 is somewhat less specific for duplexes as compared to singlestrand oligonucleotides than the E. coli enzyme. The enzyme bound tosingle strand RNA and DNA only 20 fold less well than an RNA:RNA duplexwhile the E. coli enzyme bound to single strand DNA nearly 600 fold lessthan to an RNA:RNA duplex (Table 7). The affinity of a single strandphosphorothioate oligodeoxynucleotide for both enzymes was significantrelative to the affinity for the natural substrate and accounts for theinhibition of the enzymes by members of this class oligonucleotides.Remarkably, Human RNase H1 displayed the highest affinity for a singlestrand phosphorothioate oligodeoxynucleotide. Thus, this noncleavablesubstrate is a very effective inhibitor of the enzyme and excessphosphorothioate antisense drug in cells might be highly inhibitory.

Site and Sequence Preferences for Cleavage

FIG. 2 shows the cleavage pattern for RNA duplexed with itsphosphorothioate oligodeoxynucleotide and the pattern for severalgapmers. In the parent duplex, RNA cleavage occurred at a single majorsite with minor cleavage noted at several sites 3′ to this majorcleavage site that was 8 nucleotides from 5′-terminus of the RNA. Notethat the preferred site occured at a GU dinucleotide. Cleavage ofseveral “gapmers” occurred more slowly and the major cleavage site wasat a different position from that of the parent duplex. Further, incontrast to the observations we have made for E. coli RNase H1, themajor cleavage site in gapmers treated with Human RNase H1 did not occurat the nucleotide apposed to the nucleotide adjacent to the first 2′methoxy nucleotide in the wing hybridized to the 3′ portion of the RNA.

To further evaluate the site and sequence specificities of Human RNaseH1, cleavage of substrates shown in FIG. 3 and FIG. 4 was studied. InFIG. 3, the sequence of the RNA is displayed below the sequencing gelsand the length and position of the complementary phosphodiesteroligodeoxynucleotide indicated by the solid line below the RNA sequence.This figure demonstrates several important properties of the enzyme.First, the main cleavage site was consistently observed 8-9 nucleotidesfrom the 5′-RNA:3′-DNA terminus of the duplex irrespective of whetherthere were 5′ or 3′-RNA single strand overhangs. Second, the enzyme,like E. coli RNase H1 was capable of cleaving single strand regions ofRNA adjacent to the 3′-terminus of an RNA:DNA duplex. Third, the minimumduplex length that supported any cleavage was approximately 6nucleotides. RNase protection assays were used to confirm that underconditions of the assay the shorter duplexes were fully hybridized, sothe differences observed were not due to the failure to hybridize. Toassure that the 6-nucleotide duplex was fully hybridized, the reactionswere carried out at a 50:1 DNA:RNA ratio (data not shown). Fourth, thefigure shows that for duplexes smaller than the nine base pairs, thesmaller the duplex, the slower the cleavage rate. Fifth, the preferredcleavage site was located at a GU dinucleotide.

The site and sequence specificities are further explored in FIG. 4. Thatthe enzyme displays little sequence preference is demonstrated bycomparing the rates and sites of cleavage for duplexes A, C and D. Inall cases, the preferred site of cleavage was 8-12 nucleotides from the5′-RNA:3′-DNA terminus of the duplex irrespective of the sequence.comparison of the cleavage pattern for duplexes A and B shows thatcleavage occurred at the 8-12 nucleotide position even when there wereRNA overhangs also as shown in FIG. 3. Cleavage of duplex F demonstratedthat the site of cleavage was retained even if there were 5′- and 3′-DNAoverhangs. In a longer substrate, duplex G, the main site of cleavagewas still 8-12 nucleotides from the terminus of the duplex. However,minor cleavage sites were observed throughout the RNA suggesting thatthis substrate might support binding of more than one enzyme moleculeper substrate, but that the preferred site was near the 5′-RNA:3′-DNAterminus. Finally, optimal cleavage seemed to occur when a GUdinucleotide was located 8-12 nucleotides from the 5′-RNA:3′-DNAterminus of the duplex.

To address both the mechanism of cleavage and processivity, the cleavageof 5′-labeled and 3′-labeled substrates was compared (FIG. 5). Lane Cshows that CIP treatment prior to and after digestion with Human RNaseH1 resulted in a shift in the mobility of the digested fragmentssuggesting that Human RNase H1 generates cleavage products with5′-phosphates. Thus, it is similar to E. coli RNase H1 in this regard. Asecond intriguing observation is that the addition of pC to the 3′-endof the RNA caused a shift in the position of the preferred cleavage site(A vs B or C). The four cleavage sites in the center of the duplexobserved with a 5′-phosphate labeled RNA were observed in 3′ pC-labeledsubstrates. However, the main cleavage site shifted from base pair 8 tobase pair 12. Interestingly, the sequence at both sites was GU. Thus, itis conceivable that the enzyme selects a position 8-12 nucleotide fromthe 5′-RNA:3′-DNA terminus, then cleaves at a preferred dinucleotidesuch as GU. Third, this figure considered along with the cleavagepatterns shown in FIGS. 3 and 4 demonstrates that this enzyme displaysminimal processivity in either the 5′ or 3′-direction. In no time courseexperiment using any substrate have we observed a pattern that would beconsistent with processivity. The possibility that the failure toobserve processivity in FIGS. 3 and 4 was due to processivity in the 3′to 5′-direction is excluded by the results in FIG. 5. Again, this issignificantly different from observations we have previously reportedfor E. coli RNase H1.

General Properties of Human RNase H1 Activity

The present invention also describes the properties of human RNase H1that have been characterized. As the protein studied is a his-tag fusionand was denatured and refolded, it is possible that the activity of theenzyme in its native state might be greater than we have observed.However, basic properties are certainly likely to reflect the basicproperties of the native enzyme. Numerous studies have shown that ahis-tag does not interfere with protein folding and crystallization,kinetic and catalytic properties, or nucleic acid binding propertiessince it is very small (few amino acids) and its pK is near neutral. Itis shown in the present invention that the his-tag fusion proteinbehaves like other RNase H's. It cleaved specifically the RNA strand inRNA:DNA duplexes, resulted in cleavage products with 5□-phosphatetermini (FIG. 5) and was affected by divalent cations (FIG. 1). Optimalconditions for Human RNase H1 were similar to, but not identical to, E.coli RNase H1. For the human enzyme, the Mg²⁺ optimum was 1 mM and 5 mMMg²⁺ was inhibitory. In the presence of Mg²⁺, both enzymes wereinhibited by Mn²⁺. The human enzyme was inhibited by n-ethylmaleimideand was quite stable, easily handled and did not form multimericstructures (FIG. 1). The ease of handling, denaturation, refolding andstability in various conditions suggest that the Human RNase H1 wasactive as a monomer and has a relatively stable preferred conformation.

Studies on the structure and enzymatic activities of a number of mutantsof E. coli RNase H1 have recently led to a hypothesis to explain theeffects of divalent cations termed an activation/attenuation model. Theeffects of divalent cations on Human RNase H1 are complex and areconsistent with the suggested activation/attenuation model. The aminoacids proposed to be involved in both cation binding sites are conservedin Human RNase H1.

Positional and Sequence Preferences and Processivity

The site and sequence specificity of Human RNase H1 differ substantiallyfrom E. coli RNase H1. Although neither enzyme displays significantsequence specificity and FIGS. 2-5 this manuscript, the human enzymedisplays remarkable site specificity. FIGS. 2-4 show that Human RNase H1preferentially cleaved 8-12 nucleotides 3′ from the 5′-RNA:3′-DNAterminus of a DNA:RNA duplex irrespective of whether there were 5′ or3′-RNA or DNA overhangs. The process by which a position is selected andthen within that position on the duplex a particular dinucleotide iscleaved preferentially must be relatively complex and influenced bysequence. Clearly, the dinucleotide, GU, is a preferred sequence. InFIG. 3, for example, all the duplexes contained a GU sequence near theoptimal position for the enzyme and in all cases the preferentialcleavage site was GU. Additionally, in duplexes A and B a second GU wasalso cleaved, albeit at a very slow rate. The third site in duplexes Aand B cleaved was a GG dinucleotide 7 base pairs from the 3′-RNA:5′-DNAterminus. Thus, the data suggest that the enzyme displays strongpositional preference and within the appropriate site, slight preferencefor GU dinucleotides.

The strong positional preference exhibited by Human RNase H1 suggeststhat the enzyme fixes its position on the duplex via the 5′-RNA:3′-DNAterminus. Interestingly, the in-vitro cleavage pattern observed for theenzyme is compatible with its proposed in-vivo role, namely, the removalof RNA primers during DNA replication of the lagging strand. The averagelength of the RNA primer ranges from 7-14 nucleotides. Consequently,synthesis of the lagging strand results in chimeric sequences consistingof 7-14 ribonucleotides at the 5□-terminus with contiguous stretches ofDNA extending in the 3′ direction. The positional preference observedfor Human RNase H1, (i.e., 8-12 residues from the 5′-terminus of theRNA), would suggest that cleavage of the chimeric lagging strand byRNase H1 would occur at or near the RNA:DNA junction. The removal ofresidual ribonucleatides following RNase H digestion has been shown tobe performed by the endonuclease FEN1.

FIG. 4 provides additional insight into the positional and sequencepreferences of the enzyme. When there was a GU dinucleotide present inthe correct position in the duplex, it was cleaved preferentially. Whena GU dinucleotide was absent, AU was cleaved as well as otherdinucleotides. For duplex G both a GU and a GG dinucleotide were presentwithin the preferred site, and in this case the GG dinucleotide wascleaved slightly more extensively than the GU dinucleotide. Clearly,additional duplexes of different sequences must be studied beforedefinitive conclusions concerning the roles of various sequences withinthe preferred cleavage sites can be drawn.

In FIG. 5, the 3′-terminus of the RNA was labeled with ³²pC. In thiscase the same four nucleotides were cleaved as when the RNA was 5′labeled (FIG. 5, panels B & C). However, the GU closer to the3′-terminus of the RNA was cleaved at least as rapidly as the 5′-GU.Interestingly in studies on the partially purified enzyme, differencesin the cleavage pattern were also observed when 5N-labeled substrateswere compared to 3′-labeled substrates. A possible explanation for thisobservation is that the presence of a 3′-phosphate on an oligonucleotidesubstrate affects the scanning mechanism the enzyme uses to selectpreferred positions for cleavage.

In a duplex comprised of RNA annealed to a chimeric oligonucleotide withan oligodeoxynucleotide center flanked by 2N-modified nucleotide wings,the cleavage by Human RNase H1 was directed to the DNA:RNA portion ofthe duplex as was observed for E. coli RNase H1. However, within thisregion, the preferred sites of cleavage for the human enzyme differedfrom E. coli RNase H1. E. coli RNase H1 preferentially cleaved at theribonucleotide apposed to first 2′-modified nucleotide in the wing ofantisense oligonucleotide at the 3′-end of the RNA. In contrast, thehuman enzyme preferentially cleaved at sites more centered within thegap until the gap was reduced to 5 nucleotides. Further, the minimum gapsize for the human enzyme was 5 nucleotides while that of E. coli RNaseH1 was 4 nucleotides. These differences in behavior suggest differencesin the structures of the enzymes and their interactions with substratethat will require additional study.

Although E. coli RNase H1 degrades the heteroduplex substrate in apredominantly distributive manner, the enzyme displays modest5′-3′-processivity. In contrast, Human RNase H1 evidences no 5′-3′ or3′-5′-processivity suggesting that the human enzyme hydrolyzes thesubstrate in an exclusively distributive manner. The lack ofprocessivity observed with the Human RNase H1 may be a function of thesignificantly tighter binding affinity (Table 7), thereby reducing theability of the enzyme to move on the substrate. Alternatively, HumanRNase H1 appears to fix its position on the substrate with respect tothe 5′-RNA:3′-DNA terminus and this strong positional preference maypreclude cleavage of the substrate in a processive manner. (FIG. 5).Thus, despite the fact that the enzymes are both metal-dependentendonucleases that result in cleavage products with 5′-phosphates (FIG.5) and both can cleave single-strand 3′-RNA overhangs (FIG. 5), theseenzymes display substantial differences.

E. coli RNase H1 has been suggested to exhibit “binding directionality”with respect to the RNA of the substrate such that the primary bindingregion of the enzyme is positioned several nucleotides 5′ to thecatalytic center. This results in cleavage sites being restricted fromthe 5′-RNA:3′-DNA end of a duplex, and cleavage sites occurring at the3′-RNA:5′-DNA end of the duplex and in 3′-single-strand overhangs. Thehuman enzyme behaves entirely analogously. Thus, we conclude that HumanRNase H1 likely has the same binding directionality as the E. colienzyme.

Substrate Binding

RNA:RNA duplexes have been shown to adopt an A-form conformation. Many2′-modifications shift the sugar conformation into a 3′-endo puckercharacteristic of RNA. Consequently, when hybridized to RNA, theresulting duplex is “A” form and this is manifested in a more stableduplex. 2′-F Oligonucleotides display duplex forming properties mostlike RNA, while 2′-methoxy oligonucleotides result in duplexesintermediate information between DNA:RNA and RNA:RNA duplexes.

The results shown in Table 7 demonstrate that like the E. coli enzyme,Human RNase H1 is a double strand RNA binding protein. Moreover, itdisplays some ability to discriminate between various A-form duplexes(Table 7). The observation that the Kd for an RNA:2′-F duplex is equalto that for an RNA:RNA dupex suggests that 2′-hydroxy group is notrequired for binding to the enzyme. Nevertheless, we cannot exclude thepossibility that bulkier 2′-modifications, e.g. 2′-methoxy or 2′-propylmight sterically inhibit the binding of the enzyme as well as alter theA-form quality of the duplex. The human enzyme displays substantiallygreater affinity for all oligonucleotides than the E. coli enzyme andthis is reflected in a lower Km for cleavable substrates (Tables 6 and7). In addition, the tighter binding affinity observed for Human RNaseH1 may be responsible for the 20-fold lower Vmax when compared to the E.coli enzyme. In this case, assuming that the E. coli and human enzymesexhibit similar catalytic rates (Kcat), then an increase in the bindingaffinity would result in a lower turnover rate and ultimately a lowerVmax.

The principal substrate binding site in E. coli RNase H1 is thought tobe a cluster of lysines that are believed to bind to the phosphates ofthe substrates. The interaction of the binding surface of the enzyme andsubstrate is believed to occur within the minor groove. This region ishighly conserved in the human enzyme. In addition, eukaryotic enzymescontain an extra N-terminal region of variable length containing anabundance of basic amino acids. This region is homologous with a doublestrand RNA binding motif and indeed in the S. cerevasiae RNase H hasbeen shown to bind to double strand RNA. The N-terminal extension inHuman RNase H1 is longer than that in the S. cerevasiae enzyme andappears to correspond to a more complete double strand RNA bindingmotif. Consequently, the enhanced binding of Human RNase H1 to variousnucleic acids may be due to the presence of this additional bindingsite.

As used herein, the term “alkyl” includes but is not limited to straightchain, branch chain, and cyclic unsaturated hydrocarbon groups includingbut not limited to methyl, ethyl, and isopropyl groups. Alkyl groups ofthe present invention may be substituted. Representative alkylsubstituents are disclosed in U.S. Pat. No. 5,212,295, at column 12,lines 41-50, hereby incorporated by reference in its entirety.

Alkenyl groups according to the invention are to straight chain, branchchain, and cyclic hydrocarbon groups containing at least onecarbon-carbon double bond, and alkynyl groups are to straight chain,branch chain, and cyclic hydrocarbon groups containing at least onecarbon-carbon triply bond. Alkenyl and alkynyl groups of the presentinvention can be substituted.

Aryl groups are substituted and unsubstituted aromatic cyclic moietiesincluding but not limited to phenyl, naphthyl, anthracyl, phenanthryl,pyrenyl, and xylyl groups. Alkaryl groups are those in which an arylmoiety links an alkyl moiety to a core structure, and aralkyl groups arethose in which an alkyl moiety links an aryl moiety to a core structure.

In general, the term “hetero” denotes an atom other than carbon,preferably but not exclusively N, O, or S. Accordingly, the term“heterocyclic ring” denotes a carbon-based ring system having one ormore heteroatoms (i.e., non-carbon atoms). Preferred heterocyclic ringsinclude, for example but not limited to imidazole, pyrrolidine,1,3-dioxane, piperazine, morpholine rings. As used herein, the term“heterocyclic ring” also denotes a ring system having one or more doublebonds, and one or more heteroatoms. Preferred heterocyclic ringsinclude, for example but not limited to the pyrrolidino ring.

Oligonucleotides according to the present invention that arehybridizable to a target nucleic acid preferably comprise from about 5to about 50 nucleosides. It is more preferred that such compoundscomprise from about 8 to about 30 nucleosides, with 15 to 25 nucleosidesbeing particularly preferred. As used herein, a target nucleic acid isany nucleic acid that can hybridize with a complementary nucleicacid-like compound. Further in the context of this invention,“hybridization” shall mean hydrogen bonding, which may be Watson-Crick,Hoogsteen or reversed Hoogsteen hydrogen bonding between complementarynucleobases. “Complementary” as used herein, refers to the capacity forprecise pairing between two nucleobases. For example, adenine andthymine are complementary nucleobases which pair through the formationof hydrogen bonds. “Complementary” and “specifically hybridizable,” asused herein, refer to precise pairing or sequence complementaritybetween a first and a second nucleic acid-like oligomers containingnucleoside subunits. For example, if a nucleobase at a certain positionof the first nucleic acid is capable of hydrogen bonding with anucleobase at the same position of the second nucleic acid, then thefirst nucleic acid and the second nucleic acid are considered to becomplementary to each other at that position. The first and secondnucleic acids are complementary to each other when a sufficient numberof corresponding positions in each molecule are occupied by nucleobaseswhich can hydrogen bond with each other. Thus, “specificallyhybridizable” and “complementary” are terms which are used to indicate asufficient degree of complementarity such that stable and specificbinding occurs between a compound of the invention and a target RNAmolecule. It is understood that an oligomeric compound of the inventionneed not be I 00% complementary to its target RNA sequence to bespecifically hybridizable. An oligomeric compound is specificallyhybridizable when binding of the oligomeric compound to the target RNAmolecule interferes with the normal function of the target RNA to causea loss of utility, and there is a sufficient degree of complementarityto avoid non-specific binding of the oligomeric compound to non-targetsequences under conditions in which specific binding is desired, i.e.under physiological conditions in the case of in vivo assays ortherapeutic treatment, or in the case of in vitro assays, underconditions in which the assays are performed.

As used herein, “human type 2 RNase H” and “human RNase H1” refer to thesame human RNase H enzyme. Accordingly, these terms are meant to be usedinterchangeably.

The oligonucleotides of the invention can be used in diagnostics,therapeutics and as research reagents and kits. They can be used inpharmaceutical compositions by including a suitable pharmaceuticallyacceptable diluent or carrier. They further can be used for treatingorganisms having a disease characterized by the undesired production ofa protein. The organism should be contacted with an oligonucleotidehaving a sequence that is capable of specifically hybridizing with astrand of nucleic acid coding for the undesirable protein. Treatments ofthis type can be practiced on a variety of organisms ranging fromunicellular prokaryotic and eukaryotic organisms to multicellulareukaryotic organisms. Any organism that utilizes DNA-RNA transcriptionor RNA-protein translation as a fundamental part of its hereditary,metabolic or cellular control is susceptible to therapeutic and/orprophylactic treatment in accordance with the invention. Seeminglydiverse organisms such as bacteria, yeast, protozoa, algae, all plantsand all higher animal forms, including warm-blooded animals, can betreated. Further, each cell of multicellular eukaryotes can be treated,as they include both DNA-RNA transcription and RNA-protein translationas integral parts of their cellular activity. Furthermore, many of theorganelles (e.g., mitochondria and chloroplasts) of eukaryotic cellsalso include transcription and translation mechanisms. Thus, singlecells, cellular populations or organelles can also be included withinthe definition of organisms that can be treated with therapeutic ordiagnostic oligonucleotides.

The following nonlimiting examples are provided to further illustratethe present invention.

EXAMPLES Example 1 5′-O-DMT-2′-O-(2-methoxyethyl)-5-methyl uridine and5′-O-DMT-3′-O-(2-methoxyethyl)5-methyl uridine

2′,3′-O-dibutylstannylene 5-methyl uridine (345 g) (prepared as per:Wagner et al., J. Org. Chem., 1974, 39, 24) was alkylated with2-methoxyethyl bromide (196 g) in the presence of tetrabutylammoniumiodide (235 g) in DMF (3 L) at 70° C. to give a mixture of 2′-O- and3′-O-(2-methoxyethyl)-5-methyl uridine (150 g) in nearly 1:1 ratio ofisomers. The mixture was treated with DMT chloride (110 g, DMT-Cl) inpyridine (1 L) to give a mixture of the 5′-O-DMT-nucleosides. After thestandard work-up the isomers were separated by silica gel columnchromatography. The 2′-isomer eluted first, followed by the 3′-isomer.

Example 25′-O-DMT-3′-O-(2-methoxyethyl)-5-methyl-uridine-2′-O-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite

5′-O-DMT-3′-O-(2-methoxyethyl)-5-methyluridine (5 g, 0.008 mol) wasdissolved in CH₂Cl₂ (30 mL) and to this solution, under argon,diisopropylaminotetrazolide (0.415 g) and 2-cyanoethoxy-N,N-diisopropylphosphoramidite (3.9 mL) were added. The reaction was stirred overnight.The solvent was evaporated and the residue was applied to silica columnand eluted with ethyl acetate to give 3.75 g title compound.

Example 3 5′-O-DMT-3′-O-(2-methoxyethyl)-N⁴-benzoyl-5-methyl-cytidine

5′-O-DMT-3′-O-(2-methoxyethyl)-5-methyl uridine (15 g) was treated with150 mL anhydrous pyridine and 4.5 mL of acetic anhydride under argon andstirred overnight. Pyridine was evaporated and the residue waspartitioned between 200 mL of saturated NaHCO₃ solution and 200 mL ofethylacetate. The organic layer was dried (anhydrous MgSO₄) andevaporated to give 16 g of2′-acetoxy-5′-O-(DMT)-3′-O-(2-methoxyethyl)-5-methyl uridine.

To an ice-cold solution of triazole (19.9 g) in triethylamine (50 mL)and acetonitrile (150 mL), with mechanical stirring, 9 mL of POCl₃ wasadded dropwise. After the addition, the ice bath was removed and themixture stirred for 30 min. The2′-acetoxy-5′-O-(DMT)-3′-O-(2-methoxyethyl)-5-methyl uridine (16 g in 50mL CH₃CN) was added dropwise to the above solution with the receivingflask kept at ice bath temperatures. After 2 hrs, TLC indicated a fastermoving nucleoside, C-4-triazole-derivative. The reaction flask wasevaporated and the nucleoside was partitioned between ethylacetate (500mL) and NaHCO₃ (500 mL). The organic layer was washed with saturatedNaCl solution, dried (anhydrous NgSO₄) and evaporated to give 15 g ofC-4-triazole nucleoside. This compound was then dissolved in 2:1 mixtureof NH₄OH/dioxane (100 mL:200 mL) and stirred overnight. TLC indicateddisappearance of the starting material. The solution was evaporated anddissolved in methanol to crystallize out 9.6 g of5′-O-(DMT)-3′-O-(2-methoxyethyl)5-methyl cytidine.

5′-O-DMT-3′-O-(2-methoxyethyl)-5-methyl cytidine (9.6 g, 0.015 mol) wasdissolved in 50 mL of DMF and treated with 7.37 g of benzoic anhydride.After 24 hrs of stirring, DMF was evaporated and the residue was loadedon silica column and eluted with 1:1 hexane:ethylacetate to give thedesired nucleoside.

Example 45′-O-DMT-3′-O-(2-methoxyethyl)-N⁴-benzoyl-5-methyl-cytidine-2′-O-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite

5′-O-DMT-3′-O-(2-methoxyethyl)-N⁴-benzoyl-5-methyl-cytidine-2′-O-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite was obtained from the above nucleoside using thephosphitylation protocol described for the corresponding5-methyl-uridine derivative.

Example 5 N⁶-Benzoyl-5′-O-(DMT)-3′-O-(2-methoxyethyl) adenosine

A solution of adenosine (42.74 g, 0.16 mol) in dry dimethyl formamide(800 mL) at 5° C. was treated with sodium hydride (8.24 g, 60% in oilprewashed thrice with hexanes, 0.21 mol). After stirring for 30 min,2-methoxyethyl bromide (0.16 mol) was added over 20 min. The reactionwas stirred at 5° C. for 8 h, then filtered through Celite. The filtratewas concentrated under reduced pressure followed by coevaporation withtoluene (2×100 mL). The residue was adsorbed on silica gel (100 g) andchromatographed (800 g, chloroform-methanol 9:14:1). Selected fractionswere concentrated under reduced pressure and the residue was a mixtureof 2′-O-(2-(methoxyethyl) adenosine and 3′-O-(2-methoxyethyl) adenosinein the ratio of 4:1.

The above mixture (0.056 mol) in pyridine (100 mL) was evaporated underreduced pressure to dryness. The residue was redissolved in pyridine(560 mL) and cooled in an ice water bath. Trimethylsilyl chloride (36.4mL, 0.291 mol) was added and the reaction was stirred at 5° C. for 30min. Benzoyl chloride (33.6 mL, 0.291 mol) was added and the reactionwas allowed to warm to 25° C. for 2 h and then cooled to 5° C. Thereaction was diluted with cold water (112 mL) and after stirring for 15min, concentrated ammonium hydroxide (112 Ml) was added. After 30 min,the reaction was concentrated under reduced pressure (below 30° C.)followed by coevaporation with toluene (2×100 mL). The residue wasdissolved in ethyl acetate-methanol (400 mL, 9:1) and the undesiredsilyl by-products were removed by filtration. The filtrate wasconcentrated under reduced pressure and then chromatographed on silicagel (800 g, chloroform-methanol 9:1). Selected fractions were combined,concentrated under reduced pressure and dried at 25° C./0.2 mmHg for 2 hto give pure N⁶-Benzoyl-2′-O-(2-methoxyethyl) adenosine and pureN⁶-Benzoyl-3′-O-(2-methoxyethyl) adenosine.

A solution of N⁶-Benzoyl-3′-O-(2-methoxyethyl) adenosine (11.0 g, 0.285mol) in pyridine (100 mL) was evaporated under reduced pressure to anoil. The residue was redissolved in dry pyridine (300 mL) and DMT-Cl(10.9 g, 95%, 0.31 mol) was added. The mixture was stirred at 25° C. for16 h and then poured onto a solution of sodium bicarbonate (20 g) in icewater (500 mL). The product was extracted with ethyl acetate (2×150 mL).The organic layer was washed with brine (50 mL), dried over sodiumsulfate (powdered) and evaporated under reduced pressure (below 40C).The residue was chromatographed on silica gel (400 g, ethylacetate-acetonitrile-triethylamine 99:1:195:5:1). Selected fractionswere combined, concentrated under reduced pressure and dried at 25°C./0.2 mmHg to give 16.8 g (73%) of the title compound as a foam. TheTLC was homogenous.

Example 6[N⁶-Benzoyl-5′-O-(DMT)-3′-O-(2-methoxyethyl)adenosin-2′-O-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite

The title compound was prepared in the same manner as the5-methyl-cytidine and 5-methyluridine analogs of Examples 2 and 4 bystarting with the title compound of Example 5. Purification using ethylacetate-hexanes-triethylamine 59:40:1 as the chromatography eluent gave67% yield of the title compound as a solid foam. The TLC was homogenous.³¹P-NMR (CDCl₃, H₃PO₄ std.). 147.89; 148.36 (diastereomers).

Example 7 5′-O-(DMT)-N²-isobutyryl-3′-O-(2-methoxyethyl) guanosine A.2,6-Diaminopurine riboside

To a 2 L stainless steel Parr bomb was added guanosine hydrate (100 g,0.35 mol, Aldrich), hexamethyl) disilazane (320 mL, 1.52 mol, 4.4 eq.),trimethyl) silyl triflouromethanesulfonate (8.2 mL), and toluene (350mL). The bomb was sealed and partially submerged in an oil bath (170°C.; internal T 150° C., 150 psi) for 5 days. The bomb was cooled in adry ice/acetone bath and opened. The contents were transferred withmethanol (300 mL) to a flask and the solvent was evaporated underreduced pressure. Aqueous methanol (50%, 1.2 L) was added. The resultingbrown suspension was heated to reflux for 5 h. The suspension wasconcentrated under reduced pressure to one half volume in order toremove most of the methanol. Water (600 mL) was added and the solutionwas heated to reflux, treated with charcoal (5 g) and hot filteredthrough Celite. The solution was allowed to cool to 25° C. The resultingprecipitate was collected, washed with water (200 mL) and dried at 90°C./0.2 mmHg for 5 h to give a constant weight of 87.4 g (89%) of tan,crystalline solid; mp 247° C. (shrinks), 255° C. (dec, lit. (1) mp250-252° C.); TLC homogenous (Rf 0.50, isopropanol-ammoniumhydroxide-water 16:3:1 ); PMR (DMSO), 5.73 (d, 2, 2-NH₂), 5.78 (s, 1,H-1), 6.83 (br s, 2, 6-NH₂).

B. 2′-O-(2-methoxyethyl)-2,6-diaminopurine riboside and3′-O-(2-methoxyethyl)-2,6-diaminopurine riboside

To a solution of 2,6-diaminopurine riboside (10.0 g, 0.035 mol) in drydimethyl formamide (350 mL) at 0° C. under an argon atmosphere was addedsodium hydride (60% in oil, 1.6 g, 0.04 mol). After 30 min.,2-methoxyethyl bromide (0.44 mol) was added in one portion and thereaction was stirred at 25° C. for 16 h. Methanol (10 mL) was added andthe mixture was concentrated under reduced pressure to an oil (20 g).The crude product, containing a ratio of 4:1 of the 2′/3′ isomers, waschromatographed on silica gel (500 g, chloroform-methanol 4:1). Theappropriate fractions were combined and concentrated under reducedpressure to a semi-solid (12 g). This was triturated with methanol (50mL) to give a white, hygroscopic solid. The solid was dried at 40°C./0.2 mmHg for 6 h to give a pure 2′ product and the pure 3′ isomer,which were confirmed by NMR.

C. 3′-O-2-(methoxyethyl)guanosine

With rapid stirring, 3′-O-(2-methoxyethyl)-2,6-diaminopurine riboside(0.078 mol) was dissolved in monobasic sodium phosphate buffer (0.1 M,525 mL, pH 7.3-7.4) at 25° C. Adenosine deaminase (Sigma type II, 1unit/mg, 350 mg) was added and the reaction was stirred at 25° C. for 60h. The mixture was cooled to 5° C. and filtered. The solid was washedwith water (2×25 mL) and dried at 60° C./0.2 mmHg for 5 h to give 10.7 gof first crop material. A second crop was obtained by concentrating themother liquors under reduced pressure to 125 mL, cooling to 5° C.,collecting the solid, washing with cold water (2×20 mL) and drying asabove to give 6.7 g of additional material for a total of 15.4 g (31%from guanosine hydrate) of light tan solid; TLC purity 97%.

D. N²-Isobutyryl-3′-O-2-(methoxyethyl)guanosine

To a solution of 3′-O-2-(methoxyethyl)guanosine (18.1 g, 0.0613 mol) inpyridine (300 mL) was added trimethyl silyl chloride (50.4 mL, 0.46mol). The reaction was stirred at 25° C. for 16 h. Isobutyryl chloride(33.2 mL, 0.316 mol) was added and the reaction was stirred for 4 h at25° C. The reaction was diluted with water (25 mL). After stirring for30 min, ammonium hydroxide (concentrated, 45 mL) was added until pH 6was reached. The mixture was stirred in a water bath for 30 min and thenevaporated under reduced pressure to an oil. The oil was suspended in amixture of ethyl acetate (600 mL) and water (100 mL) until a solutionformed. The solution was allowed to stand for 17 h at 25° C. Theresulting precipitate was collected, washed with ethyl acetate (2×50 mL)and dried at 60° C./0.2 mmHg for 5 h to give 16.1 g (85%) of tan solid;TLC purity 98%.

E. 5′-O-(DMT)-N′-isobutyryl-3′-O-(2-methoxyethyl) guanosine

A solution of N²-Isobutyryl-3′-O-2-(methoxyethyl) guanosine (0.051 mol)in pyridine (150 mL) was evaporated under reduced pressure to dryness.The residue was redissolved in pyridine (300 mL) and cooled to 10-15° C.DMT-C1 (27.2 g, 95%, 0.080 mol) was added and the reaction was stirredat 25° C. for 16 h. The reaction was evaporated under reduced pressureto an oil, dissolved in a minimum of methylene chloride and applied on asilica gel column (500 g). The product was eluted with a gradient ofmethylene chloride-triethylamine (99:1) to methylenechloride-methanol-triethylamine (99:1:1). Selected fractions werecombined, concentrated under reduced pressure and dried at 40° C./0.2mmHg for 2 h to afford 15 g (15.5% from guanosine hydrate) of tan foam;TLC purity 98%.

Example 8 [5′-O-(DMT)-N²-isobutyryl-3′-O-(2-methoxyethyl)guanosin-2′-O-(2-cyanoethyl-N,N-diisopropyl) phosphoramidite

The protected nucleoside from Example 7 (0.0486 mol) was placed in a dry1 L round bottom flask containing a Teflon stir-bar. The flask waspurged with argon. Anhydrous methylene chloride (400 mL) was cannulatedinto the flask to dissolve the nucleoside. Previously vacuum driedN,N-diisopropylaminohydrotetrazolide (3.0 g, 0.0174 mol) was added underargon. With stirring, bis-N,N-diisopropyl-aminocyanoethylphosphoramidite(18.8 g, 0.0689 mol) was added via syringe over 1 min (no exothermnoted). The reaction was stirred under argon at 25° C. for 16 h. Afterverifying the completion of the reaction by TLC, the reaction wastransferred to a separatory funnel (1 L). The reaction flask was rinsedwith methylene chloride (2×50 mL). The combined organic layer was washedwith saturated aq. sodium bicarbonate (200 mL) and then brine (200 mL).The organic layer was dried over sodium sulfate (50 g, powdered) for 2h. The solution was filtered and concentrated under reduced pressure toa viscous oil. The resulting phosphoramidite was purified by silica gelflash chromatography (800 g, ethyl acetate-triethylamine 99:1). Selectedfractions were combined, concentrated under reduced pressure, and driedat 25C/0.2 mmHg for 16 h to give 18.0 g (46%, 3% from guanosine hydrate)of solid foam TLC homogenous. ³¹P-NMR (CDCl₃, H₃PO₄ std.). 147.96;148.20 (diastereomers).

Example 9 5′-O-DMT-3′-O-(2-methoxyethyl)-5-methyl-uridine-2′-O-succinate

5′-O-DMT-3′-O-(2-methoxyethyl)-thymidine was first succinylated on the2′-position. Thymidine nucleoside (4 mmol) was reacted with 10.2 mLdichloroethane, 615 mg (6.14 mmol) succinic anhydride, 570 μL (4.09mmol) triethylamine, and 251 mg (2.05 mmol) 4-dimethylaminopyridine. Thereactants were vortexed until dissolved and placed in heating block at55° C. for approximately 30 minutes. Completeness of reaction checked bythin layer chromatography (TLC). The reaction mixture was washed threetimes with cold 10% citric acid followed by three washes with water. Theorganic phase was removed and dried under sodium sulfate. Succinylatednucleoside was dried under P₂O₅ overnight in vacuum oven.

Example 10 5′-O-DMT-3′-O-methoxyethyl-5-methyl-uridine-2′-O-succinoylLinked LCA CPG

5′-O-DMT-3′-O-(2-methoxyethyl)-2′-O-succinyl-thymidine was coupled tocontrolled pore glass (CPG). 1.09 g (1.52 mmol) of the succinate weredried overnight in a vacuum oven along with 4-dimethylaminopyridine(DMAP), 2,2′-dithiobis (5-nitro-pyridine) (dTNP), triphenylphosphine(TPP), and pre-acid washed CPG (controlled pore glass). After about 24hours, DMAP (1.52 mmol, 186 mg) and acetonitrile (13.7 mL) were added tothe succinate. The mixture was stirred under an atmosphere of argonusing a magnetic stirrer. In a separate flask, dTNP (1.52 mmol, 472 mg)was dissolved in acetonitrile (9.6 mL) and dichloromethane (4.1 mL)under argon. This reaction mixture was then added to the succinate. Inanother separate flask, TPP (1.52 mmol, 399 mg) was dissolved inacetonitrile (37 mL) under argon. This mixture was then added to thesuccinate/DMAP/dTNP reaction mixture. Finally, 12.23 g pre-acid washedLCA CPG (loading=115.2 μmol/g) was added to the main reaction mixture,vortexed shortly and placed on shaker for approximately 3 hours. Themixture was removed from the shaker after 3 hours and the loading waschecked. A small sample of CPG was washed with copious amounts ofacetonitrile, dichloromethane, and then with ether. The initial loadingwas found to be 63 μmol/g (3.9 mg of CPG was cleaved withtrichloroacetic acid, the absorption of released trityl cation was readat 503 nm on a spectrophotometer to determine the loading.) The wholeCPG sample was then washed as described above and dried under P₂O₅overnight in vacuum oven. The following day, the CPG was capped with 25mL CAP A (tetrahydrofuran/acetic anhydride) and 25 mL CAP B(tetrahydrofuran/pyridine/1-methyl imidazole) for approximately 3 hourson shaker. Filtered and washed with dichloromethane and ether. The CPGwas dried under P₂O₅ overnight in vacuum oven. After drying, 12.25 g ofCPG was isolated with a final loading of 90 μmol/g.

Example 11 3′-O-Methoxyethyl-5-methyl-N-benzoyl-cytidine-2′-O-succinate

5′-O-DMT-3′-O-(2-methoxy) ethyl-N-benzoyl-cytidine was firstsuccinylated on the 2′-position. Cytidine nucleoside (4 mmol) wasreacted with 10.2 mL dichloroethane, 615 mg (6.14 mmol) succinicanhydride, 570 μL (4.09 mmol) triethylamine, and 251 mg (2.05 mmol)4-dimethylaminopyridine. The reactants were vortexed until dissolved andplaced in a heating block at 55° C. for approximately 30 minutes.Completeness of reaction was checked by thin layer chromatography (TLC).The reaction mixture was washed three times with cold 10% citric acidfollowed by three washes with water. The organic phase was removed anddried under sodium sulfate. The succinylated nucleoside was dried underP₂O₅ overnight in vacuum oven.

Example 125′-O-DMT-3′-O-methoxyethyl-5-methyl-N-benzoyl-cytidine-2′-O-succinoyllinked LCA CPG

5′-O-DMT-3′-O-(2-methoxyethyl)-2′-O-succinyl-N⁴-benzoyl cytidine wascoupled to controlled pore glass (CPG). 1.05 g (1.30 mmol) of thesuccinate were dried overnight in a vacuum oven along with4-dimethylaminopyridine (DMAP), 2,2′-dithiobis (5-nitro-pyridine)(dTNP), triphenylphosphine (TPP), and pre-acid washed CPG (controlledpore glass). The following day, DMAP (1.30 mmol, 159 mg) andacetonitrile (11.7 mL) were added to the succinate. The mixture was“mixed” by a magnetic stirrer under argon. In a separate flask, dTNP(1.30 mmol, 400 mg) was dissolved in acetonitrile (8.2 mL) anddichloromethane (3.5 mL) under argon. This reaction mixture was thenadded to the succinate. In another separate flask, TPP (1.30 mmol, 338mg) was dissolved in acetonitrile (11.7 mL) under argon. This mixturewas then added to the succinate/DMAP/dTNP reaction mixture. Finally,10.46 g pre-acid washed LCA CPG (loading=115.2 μmol/g) were added to themain reaction mixture, vortexed shortly and placed on shaker forapproximately 2 hours. A portion was removed from shaker after 2 hoursand the loading was checked. A small sample of CPG was washed withcopious amounts of acetonitrile, dichloromethane, and then with ether.The initial loading was found to be 46 μmol/g. (3.4 mg of CPG werecleaved with trichloroacetic acid). The absorption of released tritylcation was read at 503 nm on a spectrophotometer to determine theloading. The whole CPG sample was then washed as described above anddried under P₂O₅ overnight in vacuum oven. The following day, the CPGwas capped with 25 mL CAP A (tetrahydrofuran/acetic anhydride) and 25 mLCAP B (tetrahydrofuran/pyridine/1-methyl imidazole) for approximately 3hours on a shaker. The material was filtered and washed withdichloromethane and ether. The CPG was dried under P₂O₅ overnight invacuum oven. After drying, 10.77 g of CPG was isolated with a finalloading of 63 μmol/g.

Example 135′-O-DMT-3′-O-methoxyethyl-N6-benzoyl-adenosine-2′-O-succinate

5′-O-DMT-3′-O-(2-methoxyethyl)-N⁶-benzoyl adenosine was firstsuccinylated on the 2′-position. 3.0 g (4.09 mmol) of the adenosinenucleoside were reacted with 10.2 mL dichloroethane, 615 mg (6.14 mmol)succinic anhydride, 570 μL (4.09 mmol) triethylamine, and 251 mg (2.05mmol) 4-dimethylaminopyridine. The reactants were vortexed untildissolved and placed in heating block at 55° C. for approximately 30minutes. Completeness of reaction was checked by thin layerchromatography (TLC). The reaction mixture was washed three times withcold 10% citric acid followed by three washes with water. The organicphase was removed and dried under sodium sulfate. Succinylatednucleoside was dried under P₂O₅ overnight in vacuum oven.

Example 145′-O-DMT-3′-O-(2-methoxyethyl)-N6-benzoyl-adenosine-2′-O-succinoylLinked LCA CPG

Following succinylation,5′-O-DMT-3′-O-(2-methoxyethyl)-2′-O-succinyl-N⁶-benzoyl adenosine wascoupled to controlled pore glass (CPG). 3.41 g (4.10 mmol) of thesuccinate were dried overnight in a vacuum oven along with4-dimethylaminopyridine (DMAP), 2,2′-dithiobis (5-nitro-pyridine)(dTNP), triphenylphosphine (TPP), and pre-acid washed CPG (controlledpore glass). The following day, DMAP (4.10 mmol, 501 mg) andacetonitrile (37 mL) were added to the succinate. The mixture was“mixed” by a magnetic stirrer under argon. In a separate flask, dTNP(4.10 mmol, 1.27g) was dissolved in acetonitrile (26 mL) anddichloromethane (11 mL) under argon. This reaction mixture was thenadded to the succinate. In another separate flask, TPP (4.10 mmol, 1.08g) was dissolved in acetonitrile (37 mL) under argon. This mixture wasthen added to the succinate/DMAP/dTNP reaction mixture. Finally, 33 gpre-acid washed LCA CPG (loading=115.2 μmol/g) were added to the mainreaction mixture, vortexed shortly and placed on shaker forapproximately 20 hours. Removed from shaker after 20 hours and theloading was checked. A small sample of CPG was washed with copiousamounts of acetonitrile, dichloromethane, and then with ether. Theinitial loading was found to be 49 μmol/g. (2.9 mg of CPG were cleavedwith trichloroacetic acid). The absorption of released trityl cation wasread at 503 nm on a spectrophotometer to determine the loading. Thewhole CPG sample was then washed as described above and dried under P₂O₅overnight in vacuum oven. The following day, the CPG was capped with 50mL CAP A (tetrahydro-furan/acetic anhydride) and 50 mL CAP B(tetrahydrofuran/pyridine/1-methyl imidazole) for approximately 1 houron the shaker. The material was filtered and washed with dichloromethaneand ether. The CPG was dried under P₂O₅ overnight in vacuum oven. Afterdrying, 33.00 g of CPG was obtained with a final loading of 66 μmol/g.

Example 155′-O-DMT-3′-O-(2-methoxyethyl)-N2-isobutyryl-guanosine-2′-O-succinate

5′-O-DMT-3′-O-(2-methoxyethy)1-N²-isobutyryl guanosine was succinylatedon the 2′-sugar position. 3.0 g (4.20 mmol) of the guanosine nucleosidewere reacted with 10.5 mL dichloroethane, 631 mg (6.30 mmol) succinicanhydride, 585 μL (4.20 mmol) triethylamine, and 257 mg (2.10 mmol)4-dimethylaminopyridine. The reactants were vortexed until dissolved andplaced in heating block at 55° C. for approximately 30 minutes.Completeness of reaction checked by thin layer chromatography (TLC). Thereaction mixture was washed three times with cold 10% citric acidfollowed by three washes with water. The organic phase was removed anddried under sodium sulfate. The succinylated nucleoside was dried underP₂O₅ overnight in vacuum oven.

Example 165′-O-DMT-3′-O-methoxyethyl-N2-isobutyryl-guanosine-2′-O-succinoyl LinkedLCA CPG

Following succinylation,5′-O-DMT-3′-O-(2-methoxyethyl)-2′-O-succinyl-N²-benzoyl guanosine wascoupled to controlled pore glass (CPG). 3.42 g (4.20 mmol) of thesuccinate were dried overnight in a vacuum oven along with4-dimethylaminopyridine (DMAP), 2,2′-dithiobis (5-nitro-pyridine)(dTNP), triphenylphosphine (TPP), and pre-acid washed CPG (controlledpore glass). The following day, DMAP (4.20 mmol, 513 mg) andacetonitrile (37.5 mL) were added to the succinate. The mixture was“mixed” by a magnetic stirrer under argon. In a separate flask, dTNP(4.20 mmol, 1.43 g) was dissolved in acetonitrile (26 mL) anddichloromethane (11 mL) under argon. This reaction mixture was thenadded to the succinate. In another separate flask, TPP (4.20 mmol, 1.10g) was dissolved in acetonitrile (37.5 mL) under argon. This mixture wasthen added to the succinate/DMAP/dTNP reaction mixture. Finally, 33.75 gpre-acid washed LCA CPG (loading=115.2 μmol/g) were added to the mainreaction mixture, vortexed shortly and placed on shaker forapproximately 20 hours. Removed from shaker after 20 hours and theloading was checked. A small sample of CPG was washed with copiousamounts of acetonitrile, dichloromethane, and then with ether. Theinitial loading was found to be 64 μmol/g. (3.4 mg of CPG were cleavedwith trichloroacetic acid). The absorption of released trityl cation wasread at 503 nm on a spectrophotometer to determine the loading. The CPGwas then washed as described above and dried under P₂O₅ overnight invacuum oven. The following day, the CPG was capped with 50 mL CAP A(tetrahydrofuran/acetic anhydride) and 50 mL CAP B(tetrahydrofuran/pyridine/1-methyl imidazole) for approximately 1 houron a shaker. The material was filtered and washed with dichloromethaneand ether. The CPG was dried under P₂O₅ overnight in vacuum oven. Afterdrying, 33.75 g. of CPG was isolated with a final loading of 72 μmol/g.

Example 17 5′-O-DMT-3′-O-[hexyl-(6-phthalimido)]-uridine

2′,3′-O-Dibutyl stannylene-uridine was synthesized according to theprocedure of Wagner et. al., J. Org. Chem., 1974, 39, 24. This compoundwas dried over P₂O₅ under vacuum for 12 hours. To a solution of thiscompound (29 g, 42.1 mmol) in 200 mL of anhydrous DMF were added (16.8g, 55 mmol) of 6-bromohexyl phthalimide and 4.5 g of sodium iodide andthe mixture was heated at 130° C. for 16 hours under argon. The reactionmixture was evaporated, co-evaporated once with toluene and the gummytar residue was applied on a silica column (500 g). The column waswashed with 2 L of EtOAc followed by eluting with 10% methanol(MeOH):90% EtOAc. The product, 2′- and 3′-isomers ofO-hexyl-6-N-phthalimido uridine, eluted as an inseparable mixture(Rf0.64 in 10% MeOH in EtOAc). By ¹³C NMR, the isomeric ration was about55% of the 2′ isomer and about 45% of the 3′ isomer. The combined yieldwas 9.2 g (46.2%). This mixture was dried under vacuum and re-evaporatedtwice with pyridine. It was dissolved in 150 mL anhydrous pyridine andtreated with 7.5 g of DMT-Cl (22.13 mmol) and 500 mg ofdimethylaminopyridine (DMAP). After 2 hours, thin layer chromatography(TLC; 6:4 EtOAc:Hexane) indicated complete disappearance of the startingmaterial and a good separation between 2′ and 3′ isomers (Rf0.29 for the2′ isomer and 0.12 for the 3′ isomer). The reaction mixture was quenchedby the addition of 5 mL of CH₃OH and evaporated under reduced pressure.The residue was dissolved in 300 mL CH₂Cl₂, washed successively withsaturated NaHCO₃ followed by saturated NaC₁ solution. It was dried overMg₂SO₄ and evaporated to give 15 g of a brown foam which was purified ona silica gel (500 g) to give 6.5 g of the 2′-isomer and 3.5 g of the 3′isomer.

Example 185′-O-DMT-3′-O-[hexyl-(6-phthalimido)]-uridine-2′-O-(2-cyanoethyl-N,N,-diisopropyl)phosphoramidite

5′-DMT-3′-O-[hexyl-(6-phthalimido)]uridine (2 g, 2.6 mmol) was dissolvedin 20 mL anhydrous CH₂Cl₂. To this solution diisopropylaminotetrazolide(0.2 g, 1.16 mmol) and 2.0 mL 2-cyanoethyl-N,N,N′,N′-tetraisopropylphosphoramidite (6.3 mmol) were added with stirred overnight. TLC (1:1EtOAc/hexane) showed complete disappearance of starting material. Thereaction mixture was transferred with CH₂Cl₂ and washed with saturatedNaHCO₃ (100 mL), followed by saturated NaCl solution. The organic layerwas dried over anhydrous Na₂SO₄ and evaporated to yield 3.8 g of a crudeproduct, which was purified in a silica column (200 g) using 1:1hexane/EtOAc to give 1.9 g (1.95 mmol, 74% yield) of the desiredphosphoramidite.

Example 19 Preparation of5′-O-DMT-3′-O-[hexyl-(6-phthalimido)]-uridine-2′-O-succinoyl-aminopropylCPG

Succinylated and capped aminopropyl controlled pore glass (CPG; 500 Åpore diameter, aminopropyl CPG, 1.0 grams prepared according to Damhaet. al., Nucl. Acids Res. 1990, 18, 3813.) was added to 12 mL anhydrouspyridine in a 100 mL round-bottom flask.1-(3-Dimethylaminopropyl)-3-ethyl-carbodiimide (DEC; 0.38 grams, 2.0mmol)], triethylamine (TEA; 100 μl, distilled over CaH₂),dimethylaminopyridine (DMAP; 0.012 grams, 0.1 mmol) and nucleoside5′-O-DMT-3′-O-[hexyl-(6-phthalimido)]uridine (0.6 grams, 0.77 mmol) wereadded under argon and the mixture shaken mechanically for 2 hours.Additional nucleoside (0.20 grams) was added and the mixture shaken for24 hours. The CPG was filtered off and washed successively withdichloromethane, triethylamine, and dichloromethane. The CPG was thendried under vacuum, suspended in 10 mL piperidine and shaken 15 minutes.The CPG was filtered off, washed thoroughly with dichloromethane andagain dried under vacuum. The extent of loading (determined byspectrophotometric assay of DMT cation in 0.3 M p-toluenesulfonic acidat 498 nm) was approximately 28 μmol/g. The5′-O-(DMT)-3′-O-[hexyl-(6-phthalimido]uridine-2′-O-succinyl-aminopropylcontrolled pore glass was used to synthesize the oligomers in an ABI380B DNA synthesizer using phosphoramidite chemistry standardconditions. A four base oligomer 5′-GACU*-3′ was used to confirm thestructure of 3′-O-hexylamine tether introduced into the oligonucleotideby NMR. As expected a multiplet signal was observed between 1.0-1.8 ppmin ¹H NMR.

Example 20 5′-O-DMT-3′-O-[hexylamino]-uridine

5′-O-(DMT)-3′-O-[hexyl-(6-phthalimido)]uridine (4.5 grams, 5.8 mmol) isdissolved in 200 mL methanol in a 500 mL flask. Hydrazine (1 ml, 31mmol) is added to the stirring reaction mixture. The mixture is heatedto 60-65° C. in an oil bath and refluxed 14 hours. The solvent isevaporated in vacuo and the residue is dissolved in dichloromethane (250mL) and extracted twice with an equal volume NH₄OH. The organic layer isevaporated to yield the crude product which NMR indicates is notcompletely pure. R_(f)0 in 100% ethyl acetate. The product is used insubsequent reactions without further purification.

Example 21 3′-O-[Propyl-(3-phthalimido)]-adenosine

To a solution of adenosine (20.0 g, 75 mmol) in dry dimethylformamide(550 ml) at 5° C. was added sodium hydride (60% oil, 4.5 g, 112 mmol).After one hour, N-(3-bromopropyl)phthalimide (23.6 g, 86 mmol) was addedand the temperature was raised to 30° C. and held for 16 hours. Ice isadded and the solution evaporated in vacuo to a gum. The gum waspartitioned between water and ethyl acetate (4×300 mL). The organicphase was separated, dried, and evaporated in vacuo and the resultantgum chromatographed on silica gel (95/5 CH₂Cl₂/MeOH) to give a whitesolid (5.7 g) of the 2′-O-(propylphthalimide)adenosine. Thee fractionscontaining the 3′-O-(propylphthalimide)adenosine were chromatographed asecond time on silica gel using the same solvent system.

Crystallization of the 2′-O-(propylphthalimide)adenosine fractions frommethanol gave a crystalline solid, m.p. 123-124C. ¹H NMR (400 MHZ:DMSO-d₆). 1.70(m, 2H, CH₂), 3.4-3.7 (m, 6H, C₅, CH₂, OCH₂, Phth CH₂),3.95 (q, 1H, C_(4′)H), 4.30 (q, 1H, C_(5′)H), 4.46 (t, 1H, C_(2′)H),5.15 (d, 1H, C_(3′)OH), 5.41 (t, 1H, C_(5′)OH), 5.95 (d, 1H, C_(1′)H)7.35 (s, 2H, NH₂), 7.8 (brs, 4H, Ar), 8.08 (s, 1H, C₂H) and 8.37 (s, 1H,C₈H). Anal. Calcd. C₂₁H₂₂N₆O₆: C, 5503; H, 4.88; N, 18.49. Found: C,55.38; H, 4.85; N, 18.46.

Crystallization of the 3′-O-(propylphthalimide)adenosine fractions fromH₂O afforded an analytical sample, m.p. 178-179C. ¹H NMR (400 MHZ:DMSO-d₆). 5.86 (d, 1H, H-1′).

Example 22 3′-O-[Propyl-(3-phthalimido)]-N6-benzoyl-adenosine

3′-O-(3-propylphthalimide)adenosine is treated with benzoyl chloride ina manner similar to the procedure of Gaffney, et al., Tetrahedron Lett.1982, 23, 2257. Purification of crude material by chromatography onsilica gel (ethyl acetate-methanol) gives the title compound.

Example 23 3′-O-[Propyl-(3-phthalimido)]-5′-O-DMT-N6-benzoyl-adenosine

To a solution of 3′-O-(propyl-3-phthalimide)-N⁶-benzoyladenosine (4.0 g)in pyridine (250 ml) is added DMT-Cl (3.3 g). The reaction is stirredfor 16 hours. The reaction is added to ice/water/ethyl acetate, theorganic layer separated, dried, and concentrated in vacuo and theresultant gum chromatographed on silica gel (ethyl acetate-methanoltriethylamine) to give the title compound.

Example 243′-O-[Propyl-(3-phthalimido)]-5′-O-DMT-N6-Benzoyl-adenosine-2′-O-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite

3′-O-(Propyl-3-phthalimide)-5′-O-DMT-N⁶-benzoyladenosine is treated with(-cyanoethoxy)chloro-N,N-diisopropyl)aminophosphane in a manner similarto the procedure of Seela, et al., Biochemistry 1987, 26, 2233.Chromatography on silica gel (EtOAc/hexane) gives the title compound asa white foam.

Example 25 3′-O-(Aminopropyl)-adenosine

A solution of 3′-O-(propyl-3-phthalimide)adenosine (8.8 g, 19 mmol), 95%ethanol (400 mL) and hydrazine (10 mL, 32 mmol) is stirred for 16 hrs atroom temperature. The reaction mixture is filtered and filtrateconcentrated in vacuo. Water (150 mL) is added and acidified with aceticacid to pH 5.0. The aqueous solution is extracted with EtOAc (2×30 mL)and the aqueous phase is concentrated in vacuo to afford the titlecompound as a HOAc salt.

Example 26 3′-O-[3-(N-trifluoroacetamido)propyl]-adenosine

A solution of 3′-O-(propylamino)adenosine in methanol (50 mL) andtriethylamine (15 mL, 108 mmol) is treated with ethyl trifluoroacetate(18 mL, 151 mmol). The reaction is stirred for 16 hrs and thenconcentrated in vacuo and the resultant gum chromatographed on silicagel (9/1, EtOAc/MeOH) to give the title compound.

Example 27 N6-Dibenzoyl-3′-O-[3-(N-trifluoroacetamido)propyl]-adenosine

3′-O-[3-(N-trifluoroacetamido)propyl]adenosine is treated as per Example22 using a Jones modification wherein tetrabutylammonium fluoride isutilized in place of ammonia hydroxide in the work up. The crude productis purified using silica gel chromatography (EtOAc/MeOH 1/1) to give thetitle compound.

Example 28N6-Dibenzoyl-5′-O-DMT-3′-O-[3-(N-trifluoroacetamido)propyl]-adenosine

DMT-Cl (3.6 g, 10.0 mmol) is added to a solution ofN⁶-(dibenzoyl)-3′-O-[3-(N-trifluoroacetamido)propyl)adenosine inpyridine (100 mL) at room temperature and stirred for 16 hrs. Thesolution is concentrated in vacuo and chromatographed on silica gel(EtOAc/TEA 99/1) to give the title compound.

Example 29N6-Dibenzoyl-5′-O-DMT-3′-O-[3-(N-trifluoroacetamido)propyl]-adenosine-2′-O-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite

A solution ofN⁶-(dibenzoyl)-5′-O-(DMT)-3′-O-[3-(N-trifluoroacetamido)-propyl]adenosinein dry CH₂Cl₂ is treated with bis-N,N-diisopropylamino cyanoethylphosphite (1.1 eqiv) and N,N-diisopropylaminotetrazolide (catalyticamount) at room temperature for 16 hrs. The reaction is concentrated invacuo and chromatographed on silica gel (EtOAc/hexane/TEA 6/4/1) to givethe title compound.

Example 30 3′-O-(butylphthalimido)-adenosine

The title compound is prepared as per Example 21, usingN-(4-bromobutyl)phthalimide in place of the 1-bromopropane.Chromatography on silica gel (EtOAC-MeOH) gives the title compound. ¹HNMR (200 MHZ, DMSO-d₆). 5.88 (d, 1H, C_(1′)H).

Example 31 N6-Benzoyl-3′-O-(butylphthalimido)-adenosine

Benzoylation of 3′-O-(butylphthalimide)adenosine as per Example 22 givesthe title compound.

Example 32 N6-Benzoyl-5′-O-DMT-3′-O-(butylphthalimido)-adenosine

The title compound is prepared from3′-O-(butyl-phthalimide)-N⁶-benzoyladenosine as per Example 22.

Example 33N6-Benzoyl-5′-O-DMT-3′-O-(butylphthalimido)-Adenosine-2′-O-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite

The title compound is prepared from3′-O-(butylphthalimide)-5′-O-DMT-N⁶-benzoyladenosine as per Example 24.

Example 34 3′-O-(Pentylphthalimido)-adenosine

The title compound is prepared as per Example 21, usingN-(5-bromopentyl)phthalimide. The crude material from the extraction ischromatographed on silica gel using CHCl₃/MeOH (95/5) to give a mixtureof the 2′ and 3′ isomers. The 2′ isomer is recrystallized from EtOH/MeOH8/2. The mother liquor is rechromatographed on silica gel to afford the3′ isomer.

2′-O-(Pentylphthalimido)adenosine: M.P. 159-160° C. Anal. Calcd. forC₂₃H₂₄N₆O₅: C, 57.26; H, 5.43; N, 17.42. Found: C, 57.03; H, 5.46; N,17.33. 3′-O-(Pentylphthalimido)adenosine: ¹H NMR (DMSO-d₆). 5.87 (d, 1H,H-1′).

Example 35 N6-Benzoyl-3′-O-(pentylphthalimido)-adenosine

Benzoylation of 3′-O-(pentylphthalimido)adenosine is achieved as per theprocedure of Example 22 to give the title compound.

Example 36 N6-Benzoyl-5′-O-DMT-3′-O-(pentylphthalimido)-adenosine

The title compound is prepared from3′-O-(pentyl-phthalimide)-N⁶-benzoyladenosine as per the procedure ofExample 23. Chromatography on silica gel (ethylacetate, hexane,triethylamine), gives the title compound.

Example 37N6-Benzoyl-5′-O-DMT-3′-O-(pentylphthalimido)-adenosine-2′-O-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite

The title compound is prepared from3′-O-(pentyl-phthalimide)-5′-O-(DMT)-N⁶-benzoyladenosine as per theprocedure of Example 24 to give the title compound.

Example 38 3′-O-(Propylphthalimido)uridine

A solution of uridine-tin complex (48.2 g, 115 mmol) in dry DMF (150 ml)and N-(3-bromopropyl)phthalimide (46 g, 172 mmol) was heated at 130° C.for 6 hrs. The crude product was chromatographed directly on silica gelCHCl₃/MeOH 95/5. The isomer ratio of the purified mixture was 2′/3′81/19. The 2′ isomer was recovered by crystallization from MeOH. Thefiltrate was rechromatographed on silica gel using CHCl₃CHCl₃/MeOH(95/5) gave the 3′ isomer as a foam.

2′-O-(Propylphthalimide)uridine: Analytical sample recrystallized fromMeOH, m.p. 165.5-166.5C, ¹NMR (200 MHZ, DMSO-d₆). 1.87 (m, 2H, CH₂),3.49-3.65 (m, 4H, C_(2′)H), 3.80-3.90 (m, 2H, C_(3′)H₁C_(4′)H), 4.09(m,1H, C_(2′)H), 5.07 (d, 1 h, C_(3′)OH), 5.16 (m, 1H, C_(5′)OH), 5.64 (d,1H, CH=), 7.84 (d, 1H, C_(1′)H), 7.92 (bs, 4H, Ar), 7.95 (d, 1H, CH═)and 11.33 (s, 1H, ArNH). Anal. C₂₀H₂₁N₃H₈, Calcd. C, 55.69; H, 4.91;, N,9.74. Found, C, 55.75; H, 5.12; N, 10.01.3′-O-(Propylphthalimide)uridine: ¹H NMR (DMSO-d₆). 5.74 (d, 1H, H-1′).

Example 39 3′-O-(Aminopropyl)-uridine

The title compound is prepared as per the procedure of Example 25.

Example 40 3′-O-[3-(N-trifluoroacetamido)propyl]-uridine

3′-O-(Propylamino)uridine is treated as per the procedure of Example 26to give the title compound.

Example 41 5′-O-DMT-3′-O-[3-(N-trifluoroacetaznido)propyl]-uridine

3′-O-[3-(N-trifluoroacetamido)propyl]uridine is treated as per theprocedure of Example 28 to give the title compound.

Example 425′-O-DMT-3′-O-[3-(N-trifluoroacetamido)propyl]-uridine-2′-O-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite

5′-O-(DMT)-3′-O-[3-(N-trifluoroacetamido)propyl]uridine is treated asper the procedure of Example 29 to give the title compound.

Example 43 3′-O-(Propylphthalimido)-cytidine

The title compounds were prepared as per the procedure of Example 21.

2′-O-(propylphthalimide)cytidine: ¹H NMR (200 MHZ, DMSO-d₆). 5.82 (d,1H, C_(1′)H).

3′-O-(propylphthalimide)cytidine: ¹H NMR (200 MHZ, DMSO-d₆). 5.72 (d,1H, C_(1′)H).

Example 44 3′-O-(Anminopropyl)-cytidine

3′-O-(Propylphthalimide)cytidine is treated as per the procedure ofExample 25 to give the title compound.

Example 45 3′-O-[3-(N-trifluoroacetamido)propyl]-cytidine

3′-O-(Propylamino)cytidine is treated as per the procedure of Example 26to give the title compound.

Example 46 N4-Benzoyl-3′-O-[3-(N-trifluoroacetamido)propyl]-cytidine

3′-O-[3-(N-trifluoroacetamido)propyl]cytidine is treated as per theprocedure of Example 27 to give the title compound.

Example 47N4-Benzoyl-5′-O-DMT-3′-O-[3-(N-trifluoroacetamido)propyl]-cytidine

N⁴-(Benzoyl)-3′-O-[3-(N-trifluoroacetamido)propyl]cytidine is treated asper the procedure of Example 28 to give the title compound.

Example 48N4-Benzoyl-5′-O-DMT-3′-O-[3-(N-trifluoroacetamido)propyl]-cytidine-2′-O-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite

N⁴-(Benzoyl)-5′-O-(DMT)-3′-O-[3-(N-trifluoroacetamido)propyl]cytidine istreated as per the procedure of Example 29 to give the title compound.

Example 49

General Procedures for Oligonucleotide Synthesis

Oligonucleotides were synthesized on a Perseptive Biosystems Expedite8901 Nucleic Acid Synthesis System. Multiple 1-μmol syntheses wereperformed for each oligonucleotide. Trityl groups were removed withtrichloroacetic acid (975 lL over one minute) followed by anacetonitrile wash. All standard amidites (0.1 M) were coupled twice percycle (total coupling time was approximately 4 minutes). All novelamidites were dissolved in dry acetonitrile (100 mg of amidite/1 mLacetonitrile) to give approximately 0.08-0.1 M solutions. Total couplingtime was approximately 6 minutes (105 μL of amidite delivered).1-H-tetrazole in acetonitrile was used as the activating agent. Excessamidite was washed away with acetonitrile. (1S)-(+)-(10-camphorsulfonyl)oxaziridine (CSO, 1.0 g CSO/8.72 mL dry acetonitrile) was used tooxidize (4 minute wait step) phosphodiester linkages while3H-1,2-benzodithiole-3-one-1,1-dioxide (Beaucage reagent, 3.4 g Beaucagereagent/200 mL acetonitrile) was used to oxidize (one minute wait step)phosphorothioate linkages. Unreacted functionalities were capped with a50:50 mixture of tetrahyrdofuran/acetic anhydride andtetrahydrofuran/pyridine/1-methyl imidazole. Trityl yields were followedby the trityl monitor during the duration of the synthesis. The finalDMT group was left intact. The oligonucleotides were deprotected in 1 mL28.0-30% ammonium hydroxide (NH₄OH) for approximately 16 hours at 55° C.Oligonucleotides were also made on a larger scale (20 μmol/synthesis).Trityl groups were removed with just over 8 mL of trichloroacetic acid.All standard amidites (0.1 M) were coupled twice per cycle (13 minutecoupling step). All novel amidites were also coupled four times percycle but the coupling time was increased to approximately 20 minutes(delivering 480 μL of amidite). Oxidation times remained the same butthe delivery of oxidizing agent increased to approximately 1.88 mL percycle. Oligonucleotides were cleaved and deprotected in 5 mL 28.0-30%NH₄OH at 55° C., for approximately 16 hours. TABLE I3′-O-(2-methoxyethyl) containing 2′-5′ linked oligonucleotides. Back-ISIS # Sequence (5′-3′)¹ bone Chemistry 17176ATG-CAT-TCT-GCC-CCC-AAG-GA* P═S 3′-O-MOE 17177ATG-CAT-TCT-GCC-CCC-AAG-G* P═S 3′-O-MOE A* 17178ATG-CAT-TCT-GCC-CCC-AAG_(o)- P═S/ 3′-O-MOE G* _(o) A* P═O 17179A*TG-CAT-TCT-GCC-CCC-AAG- P═S 3′-O-MOE GA* 17180A*TG-CAT-TCT-GCC-CCC-AAG-G* P═S 3′-O-MOE A* 17181 A*_(o)TG-CAT-TCT-GCC-AAA-AAG_(o)- P═S/ 3′-O-MOE G* _(o) A* P═O 21415A*T*G-CAT-TCT-GCC-AAA-AAG- P═S 3′-O-MOE G*A* 21416 A* _(o) T*_(o)G-CAT-TCT-GCC-AAA- P═S/ 3′-O-MOE AAG_(o)-G* _(o) A* P═O 21945 A*A*A*P═O 3′-O-MOE 21663 A*A*A*A* P═O 3′-O-MOE 20389 A*U*C*G* P═O 3′-O-MOE20390 C*G*C*-G*A*A*-T*T*C*-G*C*G* P═O 3′-O-MOE¹All nucleosides with an asterisk contain 3′-O-(2-methoxyethyl).

Example 50

General Procedure for Purification of Oligonucleotides

Following cleavage and deprotection step, the crude oligonucleotides(such as those synthesized in Example 49) were filtered from CPG usingGelman 0.45 μm nylon acrodisc syringe filters. Excess NH₄OH wasevaporated away in a Savant AS 160 automatic speed vac. The crude yieldwas measured on a Hewlett Packard 8452A Diode Array Spectrophotometer at260 nm. Crude samples were then analyzed by mass spectrometry (MS) on aHewlett Packard electrospray mass spectrometer and by capillary gelelectrophoresis (CGE) on a Beckmann P/ACE system 5000. Trityl-onoligonucleotides were purified by reverse phase preparative highperformance liquid chromatography (HPLC). HPLC conditions were asfollows: Waters 600E with 991 detector; Waters Delta Pak C4 column(7.8×300 mm); Solvent A: 50 mM triethylammonium acetate (TEA-Ac), pH7.0; B: 100% acetonitrile; 2.5 mL/min flow rate; Gradient: 5% B forfirst five minutes with linear increase in B to 60% during the next 55minutes. Larger oligo yields from the larger 20 μmol syntheses werepurified on larger HPLC columns (Waters Bondapak HC18HA) and the flowrate was increased to 5.0 mL/min. Appropriate fractions were collectedand solvent was dried down in speed vac. Oligonucleotides weredetritylated in 80% acetic acid for approximately 45 minutes andlyophilized again. Free trityl and excess salt were removed by passingdetritylated oligonucleotides through Sephadex G-25 (size exclusionchromatography) and collecting appropriate samples through a Pharmaciafraction collector. Solvent again evaporated away in speed vac. Purifiedoligonucleotides were then analyzed for purity by CGE, HPLC (flow rate:1.5 mL/min; Waters Delta Pak C4 column, 3.9×300 mm), and MS. The finalyield was determined by spectrophotometer at 260 nm. TABLE II Physicalcharacteristics of 3′-O-(2-methoxyethyl) containing 2′-5′ linkedoligonucleotides. Expected Observed HPLC² T_(R) #Ods(260 nm) Mass Mass(min.) Purified 17176 6440.743 6440.300 23.47 3006 17177 6514.8146513.910 23.67 3330 17178 6482.814 6480.900 23.06 390 17179 6513.7986513.560 23.20 3240 17180 6588.879 6588.000 23.96 3222 17181 6540.8796539.930 23.01 21415 6662.976 6662.700 24.18 4008 21416 6598.9696597.800 23.01 3060 21945 1099.924 1099.300 19.92 121 21663 1487.3241486.800 20.16 71 20389 1483.000 1482.000 62 20390 4588.000 4591.000 151²Conditions: Waters 600E with detector 991; Waters C4 column (3.9 × 300mm); Solvent A: 50 mM TEA-Ac, pH 7.0; B: 100% acetonitrile; 1.5 mL/min.flow rate; Gradient: 5% B for first five minutes with linear increase inB to 60% during the next 55 minutes.

Example 51

T_(m) Studies on Modified Oligonucleotides

Oligonucleotides synthesized in Examples 49 and 50 were evaluated fortheir relative ability to bind to their complementary nucleic acids bymeasurement of their melting temperature (T_(m)). The meltingtemperature (T_(m)), a characteristic physical property of doublehelices, denotes the temperature (in degrees centigrade) at which 50%helical (hybridized) versus coil (unhybridized) forms are present. Tm ismeasured by using the UV spectrum to determine the formation andbreakdown (melting) of the hybridization complex. Base stacking, whichoccurs during hybridization, is accompanied by a reduction in UVabsorption (hypochromicity). Consequently, a reduction in UV absorptionindicates a higher T_(m). The higher the T_(m), the greater the strengthof the bonds between the strands.

Selected test oligonucleotides and their complementary nucleic acidswere incubated at a standard concentration of 4 μM for eacholigonucleotide in buffer (100 mM NaCl, 10 mM sodium phosphate, pH 7.0,0.1 mM EDTA). Samples were heated to 90° C. and the initial absorbancetaken using a Guilford Response II Spectrophotometer (Corning). Sampleswere then slowly cooled to 15° C. and then the change in absorbance at260 nm was monitored with heating during the heat denaturationprocedure. The temperature was increased by 1 degree ° C./absorbancereading and the denaturation profile analyzed by taking the 1^(st)derivative of the melting curve. Data was also analyzed using atwo-state linear regression analysis to determine the Tm=s. The resultsof these tests for the some of the oligonucleotides from Examples 49 and50 are shown in in Table III below. TABLE III Tm Analysis ofOligonucleotides # #2′-5′ ISIS # Sequence (5′-3′) Backbone T_(m) ModsLinkages 11061 ATG-CAT-TCT-GCC-CCC-AAG-GA P═S 61.4 0 0 17176ATG-CAT-TCT-GCC-CCC-AAG-GA* P═S 61.4 1 0 17177ATG-CAT-TCT-GCC-CCC-AAG-G*A* P═S 61.3 2 1 17178ATG-CAT-TCT-GCC-CCC-AAG_(o)-G* _(o) A* P═S/P═O 61.8 2 1 17179A*TG-CAT-TCT-GCC-CCC-AAG-GA* P═S 61.1 2 1 17180A*TG-CAT-TCT-GCC-CCC-AAG-G*A* P═S 61.0 3 2 17181 A*_(o)TG-CAT-TCT-GCC-AAA-AAG_(o)-G* _(o) A* P═S/P═O 61.8 3 2 21415A*T*G-CAT-TCT-GCC-AAA-AAG-G*A* P═S 61.4 4 3 21416 A* _(o) T*_(o)G-CAT-TCT-GCC-AAA-AAG_(o)-G* _(o) A* P═S/P═O 61.7 4 3¹All nucleosides with an asterisk contain 3′-O-(2-methoxyethyl).

Example 52 NMR Experiments on Modified Oligonucleotides Comparison of3′,5′ versus, 2′,5′ Internucleotide Linkages and 2′-Substituents Versus3′-Substituents by NMR

The 400MHz ¹H spectrum of oligomer d(GAU₂*CT), whereU₂*=2′-O-aminohexyluridine showed 8 signals between 7.5 and 9.0 ppmcorresponding to the 8 aromatic protons. In addition, the anomericproton of U* appears as a doublet at 5.9 ppm with J₁′,₂′=7.5Hz,indicative of C2′-endo sugar puckering. The corresponding 2′-5′ linkedisomer shows a similar structure with J₁′,₂′=3.85 Hz at 5.75 ppm,indicating an RNA type sugar puckering at the novel modification sitefavorable for hybridization to an mRNA target. The proton spectrum ofthe oligomer d(GACU₃*), where U₃*=3′-O-hexylamine, showed the expected 7aromatic proton signals between 7.5 and 9.0 ppm and the anomeric protondoublet at 5.9 ppm with J₁′,₂′=4.4 Hz. This suggests more of a C3′-endopuckering for the 3′-O-alkylamino compounds compared to their 2′analogs. ³¹P NMR of these oligonucleotides showed the expected 4 and 3signals from the internucleotide phosphate linkages for d(GAU*CT) andd(GACU*), respectively. 3′-5′ Linked vs. 2′-5′ linked have differentretention times in RP HPLC and hence different lipophilicities, implyingpotentially different extent of interactions with cell membranes.

Example 53 T_(m) Analysis of 2′,5′-Linked Oligonucleotides Versus3′,5′-Linked Oligonucleotides

Thermal melts were done as per standarized literature procedures.Oligonucleotide identity is as follows:

Oligonucleotide A is a normal 3′-5′ linked phosphodiesteroligodeoxyribonucleotide of the sequence d(GGC TGU* CTG CG)where the *indicates the attachment site of a 2′-aminolinker. Oligonucleotide B isa normal 3′-5′ linked phosphodiester oligoribonucleotide of the sequenced(GGC TGU* CTG CG) where the * indicates the attachment site of a2′-aminolinker. Each of the ribonucleotides of the oligonucleotide,except the one bearing the * substituent, are 2′-O-methylribonucleotides. Oligonucleotide C has 2′-5′ linkage at the * positionin addition to a 3′-aminolinker at this site. The remainder of theoligonucleotide is a phosphodiester oligodeoxyribonucleotide of thesequence d(GGC TGU* CTG CG). The base oligonucleotide (no2′-aminolinker) was not included in the study. TABLE IIIa DNA RNAOLIGONUCLEOTIDE MODIFICATION TARGET TARGET A none 52.2 54.12′-aminolinker 50.9 55.5 B none 51.5 72.3 2′-aminolinker 49.8 69.3 Cnone NA 51.7 3′-aminolinker 42.7The 2′-5′ linkages demonstrated a higher melting temperature against anRNA target compared to a DNA target.

Example 54 Snake Venom Phosphodiesterase and Liver HomogenateExperiments on Oligonucleotide Stability

The following oligonucleotides were synthesized following the procedureof Example 49. TABLE IV Modified Oligonucleotides Synthesized toevaluate stability ISIS # Sequence (5′-3′)¹ Backbone Chemistry 22110TTT-TTT-TTT-TTT-TTT-T*T*T*-T* P═O 3′-O-MOE 22111 TTT-TTT-TTT-TTT-TTT-T^(#)T^(#)T^(#)-U ^(#) P═O 3′-O-MOE 22112 TTT-TTT-TTT-TTT-TTT-T*T*T*-T*P═S 3′-O-MOE 22113 TTT-TTT-TTT-TTT-TTT-T ^(#)T^(#)T^(#)-U ^(#) P═S3′-O-MOE 22114 TTT-TTT-TTT-TTT-TTT_(O)-T* _(O)T*_(O)T*_(O)T* P═S/P═O3′-O-MOE 22115 TTT-TTT-TTT-TTT-TTT_(O)-T ^(#) _(O)T^(#) _(O)T^(#) _(O)-U^(#) P═S/P═O 3′-O-MOE¹All nucleosides with an asterisk contain 3′-O-(2-methoxyethyl). Allnucleosides with a # contain 2′-O-(2-methoxyethyl).

The oligonucleotides were purified following the procedure of Example 50and analyzed for their structure. TABLE V Properties of ModifiedOligonucleotides Expected Observed HPLC² #Ods (260 nm) ISIS # Sequence(5′-3′)¹ Mass Mass T_(R) (min.) Purified 22110TTT-TTT-TTT-TTT-TTT-T*T*T*-T* 6314.189 6315.880 20.39 174 22111TTT-TTT-TTT-TTT-TTT-T ^(#)T^(#)T^(#)-U ^(#) 6004.777 5997.490 20.89 14722112 TTT-TTT-TTT-TTT-TTT-T*T*T*-T* 6298.799 6301.730 25.92 224 22113TTT-TTT-TTT-TTT-TTT-T ^(#)T^(#)T^(#)-U ^(#) 6288.745 6286.940 24.77 20922114 TTT-TTT-TTT-TTT-TTT_(O)-T* _(O)T*_(O)T*_(O)T* 6234.799 6237.15024.84 196 22115 TTT-TTT-TTT-TTT-TTT_(O)-T ^(#) _(O)T^(#) _(O)T^(#)_(O)-U ^(#) 6224.745 6223.780 23.30 340¹All nucleosides with an asterisk contain 3′-O-(2-methoxyethyl). Allnucleosides with a # contain 2′-O-(2-methoxy)ethyl.²Conditions: Waters 600E with detector 991; Waters C4 column (3.9 × 300mm); Solvent A: 50 mM TEA-Ac, pH 7.0; B: 100% acetonitrile; 1.5 mL/min.flow rate; Gradient: 5% B for first five minutes with linear increase inB to 60% during the next 55 minutes.

Example 55 3′-O-Aminopropyl Modified Oligonucleotides

Following the procedures illustrated above for the synthesis ofoligonucleotides, modified 3′-amidites were used in addition toconventional amidites to prepare the oligonucleotides listed in tablesVI and VII. Nucleosides used include:N6-benzoyl-3′-O-propylphthalimido-A-2′-amidite,2′-O-propylphthaloyl-A-3′-amidite,2′-O-methoxyethyl-thymidine-3′-amidite (RIC, Inc.),2′-O-MOE-G-3′-amidite (RI Chemical),2′-O-methoxyethyl-5-methylcytidine-3′-amidite,2′-O-methoxyethyl-adenosine-3′-amidite (RI Chemical), and5-methylcytidine-3′-amidite. 3′-propylphthalimido-A and2′-propylphthalimido-A were used as the LCA-CPG solid support. Therequired amounts of the amidites were placed in dried vials, dissolvedin acetonitrile (unmodified nucleosides were made into 1M solutions andmodified nucleosides were 100 mg/mL), and connected to the appropriateports on a Millipore Expedite™ Nucleic Acid Synthesis System. Solidsupport resin (60 mg) was used in each column for 2×1 μmole scalesynthesis (2 columns for each oligo were used). The synthesis was runusing the IBP-PS(1 μmole) coupling protocol for phosphorothioatebackbones and CSO-8 for phosphodiesters. The trityl reports indicatednormal coupling results.

After synthesis the oligonucleotides were deprotected with conc.ammonium hydroxide(aq) containing 10% of a solution of 40% methylamine(aq) at 55° C. for approximately 16 hrs. Then they were evaporated,using a Savant AS160 Automatic SpeedVac, (to remove ammonia) andfiltered to remove the CPG-resin. The crude samples were analyzed by MS,HPLC, and CE. Then they were purified on a Waters 600E HPLC system witha 991 detector using a Waters C4 Prep. scale column (Alice C4 Prep.) andthe following solvents: A: 50 mM TEA-Ac, pH 7.0 and B: acetonitrileutilizing the AMPREP2@ method. After purification the oligonucleotideswere evaporated to dryness and then detritylated with 80% acetic acid atroom temp. for approximately 30 min. Then they were evaporated.

The oligonucleotides were dissolved in conc. ammonium hydroxide and runthrough a column containing Sephadex G-25 using water as the solvent anda Pharmacia LKB SuperFrac fraction collector. The resulting purifiedoligonucleotides were evaporated and analyzed by MS, CE, and HPLC. TABLEVI Oligonucleotides bearing Aminopropyl Substituents Back- ISIS #Sequence (5′-3′)¹ bone 23185-1 A*TG-CAT-TCT-GCC-CCC-AAG-GA* P═S 23186-1A* TG-CAT-TCT-GCC-CCC-AAG-G A* P═S 23187-1 A* _(o) T _(o) G _(o)-C _(o)A _(s)T_(s)-T_(s)C_(s)T_(s)-G_(s)C_(s)C_(s)-C_(s)C_(s)C_(s)- P═S/ A _(o)A _(o) G _(o)-G _(o) A* P═O 23980-1

* _(o) T _(o) G _(o)-C _(o) A_(s)T_(s)-T_(s)C_(s)T_(s)-G_(s)C_(s)C_(s)-C_(s)C_(s)C_(s)- P═S/ A _(o) A_(o) G _(o)-G _(o)

* P═O 23981-1

* TG-CAT-TCT-GCC-CCC-AAG-G

* P═S 23982-1 A*TG-CAT-TCT-GCC-CCC-AAG-GA* P═S¹All underlined nucleosides bear a 2′-O-methoxyethyl substituent;internucleotide linkages in PS/PO oligonucleotides are indicated bysubscript >s= and >o= notations respectively; A* = 3′-aminopropyl-A;

* = 2′-aminopropyl-A; C = 5-methyl-C

TABLE VII Aminopropyl Modified Oligonucleotides Expected Observed MassMass HPLC Retention CE Retention Crude Yield Final Yield ISIS # (G/mol)(g/mol) Time (min) Time (min) (Ods) (Ods) 23185-1 6612.065 6610.5 23.195.98 948 478 23186-1 7204.697 7203.1 24.99 6.18 1075 379 23187-17076.697 7072.33 23.36 7.56 838 546 23980-1 7076.697 7102.31 23.42 7.16984 373 23981-1 7204.697 7230.14 25.36 7.18 1170 526 23982-1 6612.0656635.71 23.47 7.31 1083 463

Example 56 In Vivo Stability of Modified Oligonucleotides

The in vivo stability of selected modified oligonucleotides synthesizedin Examples 49 and 55 was determined in BALB/c mice. Following a singlei.v. administration of 5 mg/kg of oligonucleotide, blood samples weredrawn at various time intervals and analyzed by CGE. For eacholigonucleotide tested, 9 male BALB/c mice (Charles River, Wilmington,Mass.) weighing about 25 g were used. Following a one weekacclimatization the mice received a single tail-vein injection ofoligonucleotide (5 mg/kg) administered in phosphate buffered saline(PBS), pH 7.0. One retro-orbital bleed (either at 0.25, 0.5, 2 or 4 hpost-dose) and a terminal bleed (either 1, 3, 8, or 24 h post-dose) werecollected from each group. The terminal bleed (approximately 0.6-0.8 mL)was collected by cardiac puncture following ketamine/xylazineanasthesia. The blood was transferred to an EDTA-coated collection tubeand centrifuged to obtain plasma. At termination, the liver and kidneyswere collected from each mouse. Plasma and tissue homogenates were usedfor analysis to determine intact oligonucleotide content by CGE. Allsamples were immediately frozen on dry ice after collection and storedat −80C until analysis.

The CGE analysis inidcated the relative nuclease resistance of2′,5′-linked oligomers compared to ISIS 11061 (Table III, Example 51)(uniformly 2′-deoxy-phosphorothioate oligonucleotide targeted to mousec-raf). Because of the nuclease resistance of the 2′,5′-linkage, coupledwith the fact that 3′-methoxyethoxy substituents are present and affordfurther nuclease protection the oligonucleotides ISIS 17176, ISIS 17177,ISIS 17178, ISIS 17180, ISIS 17181 and ISIS 21415 were found to be morestable in plasma, while ISIS 11061 (Table III) was not. Similarobservations were noted in kidney and liver tissue. This implies that2′,5′-linkages with 3′-methoxyethoxy substituents offer excellentnuclease resistance in plasma, kidney and liver against 5′-exonucleasesand 3′-exonucleases. Thus oligonucleotides with longer durations ofaction can be designed by incorporating both the 2′,5′-linkage and3′-methoxyethoxy motifs into their structure. It was also observed that2′,5′-phosphorothioate linkages are more stable than2′,5′-phosphodiester linkages. A plot of the percentage of full lengtholigonucleotide remaining intact in plasma one hour followingadministration of an i.v. bolus of 5 mg/kg oligonucleotide is shown inFIG. 4.

A plot of the percentage of full length oligonucleotide remaining intactin tissue 24 hours following administration of an i.v. bolus of 5 mg/kgoligonucleotide is shown in FIG. 5.

CGE traces of test oligonucleotides and a standard phosphorothioateoligonucleotide in both mouse liver samples and mouse kidney samplesafter 24 hours are shown in FIG. 6. As is evident from these traces,there is a greater amount of intact oliogonucleotide for theoligonucleotides of the invention as compared to the standard seen inpanel A. The oligonucleotide shown in panel B included one substituentof the invention at each of the 5′ and 3′ ends of a phosphorothioateoligonucleotide while the phosphorothioate oligonucleotide seen in panelC included one substituent at the 5′ end and two at the 3′ end. Theoligonucleotide of panel D includes a substituent of the inventionincorporated in a 2′,5′ phosphodiester linkage at both its 5′ and 3′ends. While less stable than the oligonucleotide seen in panels B and C,it is more stable than the full phosphorothioate standardoligonucleotide of panel A.

Example 57 Control of c-raf Message in bEND Cells Using ModifiedOligonucleotides

In order to assess the activity of some of the oligonucleotides, an invitro cell culture assay was used that measures the cellular levels ofc-raf expression in bEND cells.

Cells and Reagents

The bEnd.3 cell line, a brain endothelioma, was obtained from Dr. WernerRisau (Max-Planck Institute). Opti-MEM, trypsin-EDTA and DMEM with highglucose were purchased from Gibco-BRL (Grand Island, N.Y.). Dulbecco'sPBS was purchased from Irvine Scientific (Irvine, Calif.). Sterile, 12well tissue culture plates and Facsflow solution were purchased fromBecton Dickinson (Mansfield, Mass.). Ultrapure formaldehyde waspurchased from Polysciences (Warrington, Pa.). NAP-5 columns werepurchased from Pharmacia (Uppsala, Sweden).

Oligonucleotide Treatment

Cells were grown to approximately 75% confluency in 12 well plates withDMEM containing 4.5 g/L glucose and 10% FBS. Cells were washed 3 timeswith Opti-MEM pre-warmed to 37° C. Oligonucleotide were premixed with acationic lipid (Lipofectin reagent, (GIBCO/BRL) and, serially diluted todesired concentrations and transferred on to washed cells for a 4 hourincubation at 37° C. Media was then removed and replaced with normalgrowth media for 24 hours for northern blot analysis of mRNA.

Northern Blot Analysis

For determination of mRNA levels by Northern blot analysis, total RNAwas prepared from cells by the guanidinium isothiocyanate procedure(Monia et al., Proc. Natl. Acad. Sci. USA, 1996, 93, 15481-15484) 24 hafter initiation of oligonucleotide treatment. Total RNA was isolated bycentrifugation of the cell lysates over a CsCl cushion. Northern blotanalysis, RNA quantitation and normalization to G#PDH mRNA levels weredone according to a reported procedure (Dean and McKay, Proc. Natl.Acad. Sci. USA, 1994, 91, 11762-11766). In bEND cells the2-,5-linked-3′-O-methoxyethyl oligonucleotides showed reduction of c-rafmessage activity as a function of concentration. The fact that thesemodified oligonucleotides retained activity promises reduced frequencyof dosing with these oligonucleotides which also show increased in vivonuclease resistance. All 2′,5′-linked oligonucleotides retained theactivity of parent 11061 (Table III) oligonucleotide and improved theactivity even further. A graph of the effect of the oligonucleotides ofthe present invention on c-raf expression (compared to control) in bENDcells is shown in FIG. 7.

Example 58 Synthesis of MMI-Containing Oligonucleotides

a. Bis-2′-O-methyl MMI Building Blocks

The synthesis of MMI (i.e., R=CH₃) dimer building blocks have beenpreviously described (see, e.g., Swayze, et al., Synlett 1997, 859;Sanghvi, et al., Nucleosides & Nucleotides 1997, 16 907; Swayze, et al.,Nucleosides & Nucleotides 1997, 16, 971; Dimock, et al., Nucleosides &Nucleotides 1997, 16, 1629). Generally,5′-O-(4,4′-dimethoxytrityl)-2′-O-methyl-3′-C-formyl nucleosides werecondensed with 5′-O-(N-methylhydroxylamino)-2′-O-methyl-3′-O-TBDPSnucleosides using 1 equivalent of BH₃ pyridine/1 equivalent ofpyridinium para-toluene sulfonate (PPTS) in 3:1 MeOH/THF. The resultantMMI dimer blocks were then deprotected at the lower part of the sugarwith 15 equivalents of Et₃N-2HF in THF. Thus the T*G^(iBu) dimer unitwas synthesized and phosphitylated to give T*G(MMI) phosphoramidite. Ina similar fashion, A^(BZ)*T(MMI) dimer was synthesized, succinylated andattached to controlled pore glass.

b. Oligonucleotide Synthesis

Oligonucleotides were synthesized on a Perseptive Biosystems Expedite8901 Nucleic Acid Synthesis System. Multiple 1-μmol syntheses wereperformed for each oligonucleotide. A*_(MMI)T solid support was loadedinto the column. Trityl groups were removed with trichloroacetic acid(975 μL over one minute) followed by an acetonitrile wash. Theoligonucleotide was built using a modified thioate protocol. Standardamidites were delivered (210 μL) over a 3 minute period in thisprotocol. The T*_(MMI)G amidite was double coupled using 210 μL over atotal of 20 minutes. The amount of oxidizer,3H-1,2-benzodithiole-3-one-1,1-dioxide (Beaucage reagent, 3.4 g Beaucagereagent/200 mL acetonitrile), was 225 μL (one minute wait step). Theunreacted nucleoside was capped with a 50:50 mixture oftetrahyrdofuran/acetic anhydride and tetrahydrofuran/pyridine/1-methylimidazole. Trityl yields were followed by the trityl monitor during theduration of the synthesis. The final DMT group was left intact. Afterthe synthesis, the contents of the synthesis cartridge (1 μmole) weretransferred to a Pyrex vial and the oligonucleotide was cleaved from thecontrolled pore glass (CPG) using 5 mL of 30% ammonium hydroxide (NH₄OH)for approximately 16 hours at 55° C.

c. Oligonucleotide Purification

After the deprotection step, the samples were filtered from CPG usingGelman 0.45 μm nylon acrodisc syringe filters. Excess NH₄OH wasevaporated away in a Savant AS160 automatic SpeedVac. The crude yieldwas measured on a Hewlett Packard 8452A Diode Array Spectrophotometer at260 nm. Crude samples were then analyzed by mass spectrometry (MS) on aHewlett Packard electrospray mass spectrometer. Trityl-onoligonucleotides were purified by reverse phase preparative highperformance liquid chromatography (HPLC). HPLC conditions were asfollows: Waters 600E with 991 detector; Waters Delta Pak C4 column(7.8×300 mm); Solvent A: 50 mM triethylammonium acetate (TEA-Ac), pH7.0; B: 100% acetonitrile; 2.5 mL/min flow rate; Gradient: 5% B forfirst five minutes with linear increase in B to 60% during the next 55minutes. Fractions containing the desired product (retention time=41min. for DMT-ON-16314; retention time=42.5 min. for DMT-ON-16315) werecollected and the solvent was dried off in the SpeedVac.Oligonucleotides were detritylated in 80% acetic acid for approximately60 minutes and lyophilized again. Free trityl and excess salt wereremoved by passing detritylated oligonucleotides through Sephadex G-25(size exclusion chromatography) and collecting appropriate samplesthrough a Pharmacia fraction collector. The solvent was again evaporatedaway in a SpeedVac. Purified oligonucleotides were then analyzed forpurity by CGE, HPLC (flow rate: 1.5 mL/min; Waters Delta Pak C4 column,3.9×300mm), and MS. The final yield was determined by spectrophotometerat 260 nm.

The synthesized oligonucleotides and their physical characteristics areshown, respectively, in Tables VIII and IX. All nucleosides with anasterisk contain MMI linkage. TABLE VIII ICAM-1 OligonucleotidesContaining MMI Dimers Synthesized for in Vivo Nuclease and PharmacologyStudies Back- ISIS # Sequence (5′-3′) bone 2′-Chemistry 16314 TGC ATCCCC CAG GCC P═S, Bis-2′-Ome-MMI, ACC A*T MMI 2′-H 16315 T*GC ATC CCC CAGGCC P═S, Bis-2′-Ome-MMI, ACC A*T MMI 2′-H  3082 TGC ATC CCC CAG GCG P═S2′-H, single ACC AT Mis- match 13001 TGC ATC CCC CAG GCC P═S 2′-H ACC AT

TABLE IX Physical Characteristics of MMI Oligomers Synthesized forPharmacology, and In Vivo Nuclease Studies ISIS # Retn. ExpectedObserved HPLC (min) Sequence (5′-3′) Mass (g) Mass (g) Time 16314 TGCATC CCC CAG 6295 6297 23.9 GCC ACC A*T 16315 T*G C ATC CCC CAG 6302 630324.75 GCC ACC A*T

HPLC Conditions: Waters 600E with detector 991; Waters C4 column(3.9×300 mm); Solvent A: 50 mM TEA-Ac, pH 7.0; B: 100% acetonitrile; 1.5mL/min. flow rate; Gradient: 5% B for first five minutes with linearincrease in B to 60% during the next 55 minutes.

Example 59 Synthesis of Sp Terminal Oligonucleotide

a. 3′-O-t-Butyldiphenylsilyl-thymidine (1)

5′-O-Dimethoxytritylthymidine is silylated with 1 equivalent oft-butyldiphenylsilyl chloride (TBDPSCl) and 2 equivalents of imidazolein DMF solvent at room temperature. The 5′-protecting group is removedby treating with 3% dichloracetic acid in CH₂Cl₂.

b. 5′-O-Dimethoxytrityl-thymidin-3′-O-yl-N,N-diisopropylamino(S-pivaloyl-2-mercaptoethoxy) phosphoramidite (2)

5′-O-Dimethoxytrityl thymidine is treated withbis-(N,N-diisopropylamino)-S-pivaloyl-2-mercaptoethoxy phosphoramiditeand tetrazole in CH₂Cl₂/CH₃CN as described by Guzaev et al., Bioorganic& Medicinal Chemistry Letters 1998, 8, 1123) to yield the titlecompound.

c. 5′-O-Dimethoxytrityl-2′-deoxy-adenosin-3′-O-yl-N,N-diisopropylamino(S-pivaloyl-2-mercapto ethoxy) phosphoramidite (3)

5′-O-Dimethoxytrityl-N-6-benzoyl-2′-deoxy-adenosine is phosphitylated asin the previous example to yield the desired amidite.

d. 3′-O-t-Butyldiphenylsilyl-2′-deoxy-N₂-isobutyryl-guanosine (4)

5′-O-Dimethoxytrityl-2′-deoxy-N₂-isobutyryl-guanisine is silylated withTBDPSCl and imidazole in DMF. The 5′-DMT is then removed with 3% DCA inCH₂Cl₂.

e. T_((Sp))G Dimers and T_((S)) Phosphoramidite

Compounds 4 and 2 are condensed (1:1 equivalents) using 1H-tetrazole inCH₃CN solvent followed by sulfurization employing Beaucage reagent (see,e.g., Iyer, et al., J. Org. Chem. 1990, 55, 4693). The dimers (TG) areseparated by column chromatography and the silyl group is deprotectedusing t-butyl ammonium fluoride/THF to give Rp and Sp dimers of T_(s)G.Small amounts of these dimers are completely deprotected and treatedwith either P1 nuclease or snake venom phosphodiesterase. The R isomeris resistant to P1 nuclease and hydrolyzed by SVPD. The S isomer isresistant to SVPD and hydrolyzed P1 nuclease. The Sp isomer of the fullyprotected T_(s)G dimer is phosphitylated to giveDMT-T-Sp-G-phosphoramidite.

f. A,T Dimers and Solid Support Containing A_(SP)T Dimer

Compounds 3 and 1 are condensed using 1H-tetrazole in CH₃CN solventfollowed by sulfurization to give AT dimers. The dimers are separated bycolumn chromatography and the silyl group is deprotected with TBAF/THF.The configurational assignments are done generally as in the previousexample. The Sp isomer is then attached to controlled pore glassaccording to standard procedures to give DMT-A_(SP)-T-CPGoligomerization with chirally pure Sp dimer units at the termini.

g. Oligonucleotide Synthesis

The oligonucleotide having the sequence T*GC ATC CCC CAG GCC ACC A*T issynthesized, where T*G and A*T represent chiral Sp dimer blocksdescribed above. DMT-A_(SP)-T-CPG is taken in the synthesis column andthe next 16b residues are built using standard phosphorothioateprotocols and 3H-1,2-benzodithiol-3-one 1,1 dioxide as the sulfurizingagent. After building this 18 mer unit followed by final detritylation,the chiral Sp dimer phosphoramidite of 5′-DMT-Tsp-G amidite is coupledto give the desired antisense oligonucleotide. This compound is thendeprotected in 30% NH₄OH over 16 hours and the oligomer purified in HPLCand desalted in Sephader G-25 column. The final oligomer has Spconfiguration at the 5′-terminus and 3′-terminus and the interior hasdiastereomeric mixture of Rp and Sp configurations.

Example 60 Evaluation of in Vivo Stability of MMI CappedOligonucleotides Mouse Experiment Procedures

For each oligonucleotide tested, 9 male BALB/c mice (Charles River,Wilmington, Mass.), weighing about 25 g was used (Crooke et al., J.Pharmacol. Exp. Ther., 1996, 277, 923). Following a 1-week acclimation,mice received a single tail vein injection of oligonucleotide (5 mg/kg)administered in phosphate buffered saline (PBS), pH 7.0 Oneretro-orbital bleed (either 0.25, 0.5, 2 or 41 v post dose) and aterminal bleed (either 1, 3, 8 or 24 h post dose) were collected fromeach group. The terminal bleed (approximately 0.6-0.8 mL) was collectedby cardiac puncture following ketamine/xylazine anesthesia. The bloodwas transferred to an EDTA-coated collection tube and centrifuged toobtain plasma. At termination, the liver and kidneys were collected fromeach mouse. Plasma and tissues homogenates were used for analysis fordetermination of intact oligonucleotide content by CGE. All samples wereimmediately frozen on dry ice after collection and stored at −80° C.until analysis.

The capillary gel electrophoretic analysis indicated the relativenuclease resistance of MMI capped oligomers compared to ISIS 3082(uniform 2′-deoxy phosphorothioate). Because of the resistance of MMIlinkage to nucleases, the compound 16314 was found to be stable inplasma while 3082 was not. However, in kidney and liver, the compound16314 also showed certain amount of degradation. This implied that while3′-exonuclease is important in plasma, 5′-exonucleases or endonucleasesmay be active in tissues. To distinguish between these twopossibilities, the data from 16315 was analyzed. In plasma as well as intissues, (liver and kidney) the compound was stable in various timepoints. (1, 3 and 24 hrs.). The fact that no degradation was detectedproved that 5′-exonucleases and 3′-exonuclease are prevalent in tissuesand endonucleases are not active. Furthermore, a single linkage (MMI orSp thioate linkage) is sufficient as a gatekeeper against nucleases.

Control of ICAM-1 Expression Cells and Reagents

The bEnd.3 cell line, a brain endothelioma, was the kind gift of Dr.Werner Risau (Max-Planck Institute). Opti-MEM, trypsin-EDTA and DMEMwith high glucose were purchased from Gibco-BRL (Grand Island, N.Y.).Dulbecco's PBS was purchased from Irvine Scientific (Irvine, Calif.).Sterile, 12 well tissue culture plates and Facsflow solution werepurchased from Becton Dickinson (Mansfield, Mass.). Ultrapureformaldehyde was purchased from Polysciences (Warrington, Pa.).Recombinant human TNF-a was purchased from R&D Systems (Minneapolis,Minn.). Mouse interferon. was purchased from Genzyme (Cambridge, Mass.).Fraction V, BSA was purchased from Sigma (St. Louis, Mo.). The mouseICAM-1-PE, VCAM-1-FITC, hamster IgG-FITC and rat IgG_(2a)-PE antibodieswere purchased from Pharmingen (San Diego, Calif.). Zeta-Probe nylonblotting membrane was purchased from Bio-Rad (Richmond, Calif.).QuickHyb solution was purchased from Stratagene (La Jolla, Calif.). AcDNA labeling kit, Prime-a-Gene, was purchased from ProMega (Madison,WI). NAP-5 columns were purchased from Pharmacia (Uppsala, Sweden).

Oligonucleotide Treatment

Cells were grown to approximately 75% confluency in 12 well plates withDMEM containing 4.5 g/L glucose and 10% FBS. Cells were washed 3 timeswith Opti-MEM pre-warmed to 37° C. Oligonucleotide was premixed withOpti-MEM, serially diluted to desired concentrations and transferredonto washed cells for a 4 hour incubation at 37° C. Media was removedand replaced with normal growth media with or without 5 ng/mL TNF- and200 U/mL interferon, incubated for 2 hours for northern blot analysis ofmRNA or overnight for flow cytometric analysis of cell surface proteinexpression.

Flow Cytometry

After oligonucleotide treatment, cells were detached from the plateswith a short treatment of trypsin-EDTA (1-2 min.). Cells weretransferred to 12×75 mm polystyrene tubes and washed with 2% BSA, 0.2%sodium azide in D-PBS at 4° C. Cells were centrifuged at 1000 rpm in aBeckman GPR centrifuge and the supernatant was then decanted. ICAM-1,VCAM-1 and the control antibodies were added at 1 ug/mL in 0.3 mL of theabove buffer. Antibodies were incubated with the cells for 30 minutes at4° C. in the dark, under gentle agitation. Cells were washed again asabove and then resuspended in 0.3 mL of FacsFlow buffer with 0.5%ultrapure formaldehyde. Cells were analyzed on a Becton DickinsonFACScan. Results are expressed as percentage of control expression,which was calculated as follows: [((CAM expression foroligonucleotide-treated cytokine induced cells)−(basal CAMexpression))/((cytokine-induced CAM expression)−(basal CAMexpression))]×100. For the experiments involving cationic lipids, bothbasal and cytokine-treated control cells were pretreated with Lipofectinfor 4 hours in the absence of oligonucleotides.

The results reveal the following: 1) Isis 3082 showed an expected doseresponse (25-200 nM); 2) Isis 13001 lost its ability to inhibit ICAM-1expression as expected from a mismatch compound, thus proving anantisense mechanism; 3) 3′-MMI capped oligomer 16314 improved theactivity of 3082, and at 200 nM concentration, nearly twice as active as3082; 4) 5′- and 3′-MMI capped oligomer is the most potent compound andit is nearly 4 to 5 times more efficacious than the parent compound at100 and 200 nM concentrations. Thus, improved nuclease resistanceincreased the potency of the antisense oligonucleotides.

Example 61 Control of H-ras Expression

Antisense oligonucleotides targeting the H-ras message were tested fortheir ability to inhibit production of H-ras mRNA in T-24 cells. Forthese test, T-24 cells were plated in 6-well plates and then treatedwith various escalating concentrations of oligonucleotide in thepresence of cationic lipid (Lipofectin, GIBCO) at the ratio of 2.5 μg/mlLipofectin per 100 nM oligonucleotide. Oligonucleotide treatment wascarried out in serum free media for 4 hours. Eighteen hours aftertreatment the total RNA was harvested and analyzed by northern blot forH-ras mRNA and control gene G3PDH. The data is presented in FIGS. 8 and9 in bar graphs as percent control normalized for the G3PDH signal. Ascan be seen, the oligonucleotide having a single MMI linkage in each ofthe flank regions showed significant reduction of H-ras mRNA.

Example 62 5-Lipoxygenase Analysis and Assays

A. Therapeutics

For therapeutic use, an animal suspected of having a diseasecharacterized by excessive or abnormal supply of 5-lipoxygenase istreated by administering a compound of the invention. Persons ofordinary skill can easily determine optimum dosages, dosingmethodologies and repetition rates. Such treatment is generallycontinued until either a cure is effected or a diminution in thediseased state is achieved. Long term treatment is likely for somediseases.

B. Research Reagents

The oligonucleotides of the invention will also be useful as researchreagents when used to cleave or otherwise modulate 5-lipoxygenase mRNAin crude cell lysates or in partially purified or wholly purified RNApreparations. This application of the invention is accomplished, forexample, by lysing cells by standard methods, optimally extracting theRNA and then treating it with a composition at concentrations ranging,for instance, from about 100 to about 500 ng per 10 Mg of total RNA in abuffer consisting, for example, of 50 mm phosphate, pH ranging fromabout 4-10 at a temperature from about 30 to about 50° C. The cleaved5-lipoxygenase RNA can be analyzed by agarose gel electrophoresis andhybridization with radiolabeled DNA probes or by other standard methods.

C. Diagnostics

The oligonucleotides of the invention will also be useful in diagnosticapplications, particularly for the determination of the expression ofspecific mRNA species in various tissues or the expression of abnormalor mutant RNA species. In this example, while the macromolecules targeta abnormal mRNA by being designed complementary to the abnormalsequence, they would not hybridize to normal mRNA. Tissue samples can behomogenized, and RNA extracted by standard methods. The crude homogenateor extract can be treated for example to effect cleavage of the targetRNA. The product can then be hybridized to a solid support whichcontains a bound oligonucleotide complementary to a region on the 5′side of the cleavage site. Both the normal and abnormal 5′ region of themRNA would bind to the solid support. The 3′ region of the abnormal RNA,which is cleaved, would not be bound to the support and therefore wouldbe separated from the normal mRNA.

Targeted mRNA species for modulation relates to 5-lipoxygenase; however,persons of ordinary skill in the art will appreciate that the presentinvention is not so limited and it is generally applicable. Theinhibition or modulation of production of the enzyme 5-lipoxygenase isexpected to have significant therapeutic benefits in the treatment ofdisease. In order to assess the effectiveness of the compositions, anassay or series of assays is required.

D. In Vitro Assays

The cellular assays for 5-lipoxygenase preferably use the humanpromyelocytic leukemia cell line HL-60. These cells can be induced todifferentiate into either a monocyte like cell or neutrophil like cellby various known agents. Treatment of the cells with 1.3% dimethylsulfoxide, DMSO, is known to promote differentiation of the cells intoneutrophils. It has now been found that basal HL-60 cells do notsynthesize detectable levels of 5-lipoxygenase protein or secreteleukotrienes (a downstream product of 5-lipoxygenase). Differentiationof the cells with DMSO causes an appearance of 5-lipoxygenase proteinand leukotriene biosynthesis 48 hours after addition of DMSO. Thusinduction of 5-lipoxygenase protein synthesis can be utilized as a testsystem for analysis of oligonucleotides which interfere with5-lipoxygenase synthesis in these cells.

A second test system for oligonucleotides makes use of the fact that5-lipoxygenase is a “suicide” enzyme in that it inactivates itself uponreacting with substrate. Treatment of differentiated HL-60 or othercells expressing 5 lipoxygenase, with 10 μM A23187, a calcium ionophore,promotes translocation of 5-lipoxygenase from the cytosol to themembrane with subsequent activation of the enzyme. Following activationand several rounds of catalysis, the enzyme becomes catalyticallyinactive. Thus, treatment of the cells with calcium ionophoreinactivates endogenous 5-lipoxygenase. It takes the cells approximately24 hours to recover from A23187 treatment as measured by their abilityto synthesize leukotriene B₄. Macromolecules directed against5-lipoxygenase can be tested for activity in two HL-60 model systemsusing the following quantitative assays. The assays are described fromthe most direct measurement of inhibition of 5-lipoxygenase proteinsynthesis in intact cells to more downstream events such as measurementof 5-lipoxygenase activity in intact cells.

A direct effect which oligonucleotides can exert on intact cells andwhich can be easily be quantitated is specific inhibition of5-lipoxygenase protein synthesis. To perform this technique, cells canbe labeled with ³⁵S-methionine (50 μCi/mL) for 2 hours at 37° C. tolabel newly synthesized protein. Cells are extracted to solubilize totalcellular proteins and 5-lipoxygenase is immunoprecipitated with5-lipoxygenase antibody followed by elution from protein A Sepharosebeads. The immunoprecipitated proteins are resolved bySDS-polyacrylamide gel electrophoresis and exposed for autoradiography.The amount of immunoprecipitated 5-lipoxygenase is quantitated byscanning densitometry.

A predicted result from these experiments would be as follows. Theamount of 5-lipoxygenase protein immunoprecipitated from control cellswould be normalized to 100%. Treatment of the cells with 1 μM, 10 μM,and 30 μM of the macromolecules of the invention for 48 hours wouldreduce immunoprecipitated 5-lipoxygenase by 5%, 25% and 75% of control,respectively.

Measurement of 5-lipoxygenase enzyme activity in cellular homogenatescould also be used to quantitate the amount of enzyme present which iscapable of synthesizing leukotrienes. A radiometric assay has now beendeveloped for quantitating 5-lipoxygenase enzyme activity in cellhomogenates using reverse phase HPLC. Cells are broken by sonication ina buffer containing protease inhibitors and EDTA. The cell homogenate iscentrifuged at 10,000×g for 30 min and the supernatants analyzed for5-lipoxygenase activity. Cytosolic proteins are incubated with 10 μM¹⁴C-arachidonic acid, 2 mM ATP, 50 μM free calcium, 100 μg/mLphosphatidylcholine, and 50 mM bis-Tris buffer, pH 7.0, for 5 min at 37°C. The reactions are quenched by the addition of an equal volume ofacetone and the fatty acids extracted with ethyl acetate. The substrateand reaction products are separated by reverse phase HPLC on a NovapakC18 column (Waters Inc., Millford, Mass.). Radioactive peaks aredetected by a Beckman model 171 radiochromatography detector. The amountof arachidonic acid converted into di-HETE's and mono-HETE's is used asa measure of 5-lipoxygenase activity.

A predicted result for treatment of DMSO differentiated HL-60 cells for72 hours with effective the macromolecules of the invention at 1 μM, 10μM, and 30 μM would be as follows. Control cells oxidize 200 μmolarachidonic acid/5 min/10⁶ cells. Cells treated with 1 μM, 10 μM, and 30μM of an effective oligonucleotide would oxidize 195 pmol, 140 pmol, and60 pmol of arachidonic acid/5 min/10⁶ cells respectively.

A quantitative competitive enzyme linked immunosorbant assay (ELISA) forthe measurement of total 5-lipoxygenase protein in cells has beendeveloped. Human 5-lipoxygenase expressed in E. coli and purified byextraction, Q-Sepharose, hydroxyapatite, and reverse phase HPLC is usedas a standard and as the primary antigen to coat microtiter plates.Purified 5-lipoxygenase (25 ng) is bound to the microtiter platesovernight at 4° C. The wells are blocked for 90 min with 5% goat serumdiluted in 20 mM Tris!HCL buffer, pH 7.4, in the presence of 150 mM NaCl(TBS). Cell extracts (0.2% Triton X-100, 12,000×g for 30 min.) orpurified 5-lipoxygenase were incubated with a 1:4000 dilution of5-lipoxygenase polyclonal antibody in a total volume of 100 μL in themicrotiter wells for 90 min. The antibodies are prepared by immunizingrabbits with purified human recombinant 5-lipoxygenase. The wells arewashed with TBS containing 0.05% tween 20 (TBST), then incubated with100 μL of a 1:1000 dilution of peroxidase conjugated goat anti-rabbitIgG (Cappel Laboratories, Malvern, Pa.) for 60 min at 25° C. The wellsare washed with TBST and the amount of peroxidase labeled secondantibody determined by development with tetramethylbenzidine.

Predicted results from such an assay using a 30 mer oligonucleotide at 1μM, 10 μM, and 30 μM would be 30 ng, 18 ng and 5 ng of 5-lipoxygenaseper 10⁶ cells, respectively with untreated cells containing about 34 ng5-lipoxygenase.

A net effect of inhibition of 5-lipoxygenase biosynthesis is adiminution in the quantities of leukotrienes released from stimulatedcells. DMSO-differentiated HL-60 cells release leukotriene B4 uponstimulation with the calcium ionophore A23187. Leukotriene B4 releasedinto the cell medium can be quantitated by radioimmunoassay usingcommercially available diagnostic kits (New England Nuclear, Boston,Mass.). Leukotriene B4 production can be detected in HL-60 cells 48hours following addition of DMSO to differentiate the cells into aneutrophil-like cell. Cells (2×10⁵ cells/mL) will be treated withincreasing concentrations of the macromolecule for 48-72 hours in thepresence of 1.3% DMSO. The cells are washed and resuspended at aconcentration of 2×10⁶ cell/mL in Dulbecco's phosphate buffered salinecontaining 1% delipidated bovine serum albumin. Cells are stimulatedwith 10 μM calcium ionophore A23187 for 15 min and the quantity of LTB4produced from 5×10⁵ cell determined by radioimmunoassay as described bythe manufacturer.

Using this assay the following results would likely be obtained with anoligonucleotide directed to the 5-LO mRNA. Cells will be treated for 72hours with either 1 μM, 10 μM or 30 μM of the macromolecule in thepresence of 1.3% DMSO. The quantity of LTB₄ produced from 5×10⁵ cellswould be expected to be about 75 pg, 50 pg, and 35 pg, respectively withuntreated differentiated cells producing 75 pg LTB₄.

E. In Vivo Assay

Inhibition of the production of 5-lipoxygenase in the mouse can bedemonstrated in accordance with the following protocol. Topicalapplication of arachidonic acid results in the rapid production ofleukotriene B₄, leukotriene C₄ and prostaglandin E₂ in the skin followedby edema and cellular infiltration. Certain inhibitors of 5-lipoxygenasehave been known to exhibit activity in this assay. For the assay, 2 mgof arachidonic acid is applied to a mouse ear with the contralateral earserving as a control. The polymorphonuclear cell infiltrate is assayedby myeloperoxidase activity in homogenates taken from a biopsy 1 hourfollowing the administration of arachidonic acid. The edematous responseis quantitated by measurement of ear thickness and wet weight of a punchbiopsy. Measurement of leukotriene B₄ produced in biopsy specimens isperformed as a direct measurement of 5-lipoxygenase activity in thetissue. Oligonucleotides will be applied topically to both ears 12 to 24hours prior to administration of arachidonic acid to allow optimalactivity of the compounds. Both ears are pretreated for 24 hours witheither 0.1 μmol, 0.3 μmol, or 1.0 μmol of the macromolecule prior tochallenge with arachidonic acid. Values are expressed as the mean forthree animals per concentration. Inhibition of polymorphonuclear cellinfiltration for 0.1 μmol, 0.3 μmol, and 1 μmol is expected to be about10%, 75% and 92% of control activity, respectively. Inhibition of edemais expected to be about 3%, 58% and 90%, respectively while inhibitionof leukotriene B₄ production would be expected to be about 15%, 79% and99%, respectively.

Example 63 5′-O-DMT-2′-deoxy-2′-methylene-5-methyluridine-3′-(2-cyanoethyl-N,N-dilsoproppyl) phosphorarmidite

2′-Deoxy-2′-methylene-3′,5′-O-(tetraisopropyldisiloxane-1,3,diyl)-5-methyl uridine is synthesized following theprocedures reported for the corresponding uridine derivative (Hansske,F.; Madej, D.; Robins, M. J. Tetrahedron (1984) 40, 125; Matsuda, A.;Takenusi, K.;, Tanaka, S.; Sasaki, T.; Ueda, T. J. Med. Chem. (1991) 34,812; See also Cory, A. H.; Samano, V.; Robins, M. J.; Cory, J. G.2′-Deoxy-2′-methylene derivatives of adenosine, guanosine, tubercidin,cytidine and uridine as inhibitors of L1210 cell growth in culture.Biochem. Pharmacol. (1994), 47(2), 365-71.)

It is treated with IM TBAF in THF to give 2′-deoxy-2′-methylene-5-methyluridine. It is dissolved in pyridine and treated with DMT-Cl and stirredto give the 5′-O-DMT-2′-deoxy-2′-methylene-5-methyl uridine. Thiscompound is treated with 2-cyanoethyl-N,N-diisopropyl phosphoramiditeand diisopropylaminotetrazolide. In a similar manner the correspondingN-6 benzoyl adenosine, N-4-benzoyl cytosine, N-2-isobutyryl guanosinephosphoramidite derivatives are synthesized.

Example 63 Synthesis of 3′-O-4′-C-methyleneribonucleoside

5′-O-DMT-3′-O-4′-C-methylene uridine and 5-methyl uridine aresynthesized and phosphitylated according to the procedure of Obika etal. (Obika et al. Bioorg. Med. Chem. Lett. (1999) 9, 515-158). Theamidites are incorporated into oligonucleotides using the protocolsdescribed above.

Example 64 Synthesis of 2′-methylene phosphoramidites

5′-O-DMT-2′-(methyl)-3′-O-(2-cyanoethyl-N,N-diisopropylamine)-5-methyluridine-phosphoramidite,5′-O-DMT-2′-(methyl)-N-6-benzoyl adenosine(3′-O-2-cyanoethyl-N,N-diisopropylamino) phosphoramidite,5′-O-DMT-2′-(methyl)-N2-isoburytylguanosine-3′-O-(2-cyanoethyl-N,N-diisopropylamino) phosphoramidite and5′-O-DMT-2′-(methyl)-N-4-benzoylcytidine-3′-O-(2-cyanoethyl-N,N-diisopropylamino) phosphoramidites wereobtained by the phosphitylation of the corresponding nucleosides. Thenucleosides were synthesized according to the procedure described byIribarren, Adolfo M.; Cicero, Daniel O.; Neuner, Philippe J. Resistanceto degradation by nucleases of(2′S)-2′-deoxy-2′-C-methyloligonucleotides, novel potential antisenseprobes. Antisense Res. Dev., (1994), 4(2), 95-8; Schmit, Chantal;Bevierre, Marc-Olivier; De Mesmaeker, Alain; Altmann, Karl-Heinz. “Theeffects of 2′- and 3′-alkyl substituents on oligonucleotidehybridization and stability”. Bioorg. Med. Chem. Lett. (1994), 4(16),1969-74.

The phosphitylation is carried out by using the bisamidite procedure.

Example 65 Synthesis of 2′-S-methyl phosphoramidites

5′-O-DMT-2′-S-(methyl)-3′-O-(2-cyanoethyl-N,N-diisopropylamine)-5-methyluridine-phosphoramidite, 5′-O-DMT-2′-S(methyl)-N-6-benzoyl adenosine(3′-O-2-cyanoethyl-N,N-diisopropylamino) phosphoramidite,5′-O-DMT-2′-S-(methyl)-N2-isoburytylguanosine-3′-O-(2-cyanoethyl-N,N-diisopropylamino) phosphoramidite and5′-O-DMT-2′-S-(methyl)-N-4-benzoylcytidine-3′-O-(2-cyanoethyl-N,N-diisopropylamino) phosphoramidites wereobtained by the phosphitylation of the corresponding nucleosides. Thenucleosides were synthesized according to the procedure described byFraser et al. (Fraser, A.; Wheeler, P.; Cook, P. D.; Sanghvi, Y. S. J.Heterocycl. Chem. (1993) 31, 1277-1287). The phosphitylation is carriedout by using the bisamidite procedure.

Example 66 Synthesis of 2′-O-methy-β-D-arabinofuranosyl compounds

2′-O-Methyl-β-D-arabinofuranosyl-thymidine containing oligonucleotideswere synthesized following the procedures of Gotfredson et. al.(Gotfredson, C. H. et. al. Tetrahedron Lett. (1994) 35,6941-6944;Gotfredson, C. H. et. al. Bioorg. Med. Chem. (1996) 4,1217-1225).5′-O-DMT-2′-ara-(O-methyl)-3′-O-(2-cyanoethyl-N,N-diisopropylamine)-5-methyluridine-phosphoramidite, 5′-O-DMT-2′-ara-(O-methyl)-N-6-benzoyladenosine (3′-O-2-cyanoethyl-N,N-diisopropylamino) phosphoramidite,5′-O-DMT-2′-ara-(O-methyl)-N2-isoburytylguanosine-3′-O-(2-cyanoethyl-N,N-diisopropylamino) phosphoramidite and5′-O-DMT-2′-ara-(O-methyl)-N-4-benzoylcytidine-3′-O-(2-cyanoethyl-N,N-diisopropylamino) phosphoramidites areobtained by the phosphitylation of the corresponding nucleosides. Thenucleosides are synthesized according to the procedure described byGotfredson, C. H. et. al. Tetrahedron Lett. (1994) 35, 6941-6944;Gotfredson, C. H. et. al. Bioorg. Med. Chem. (1996) 4, 1217-1225. Thephosphitylation is carried out by using the bisamidite procedure.

Example 67 Synthesis of 2′-fluoro-β-D-arabinofuranosyl compounds

2′-Fluoro-β-D-arabinofuranosyl oligonucleotides are synthesizedfollowing the procedures by Kois,P. et al., Nucleosides Nucleotides 12,1093, 1993 and Damha et al., J. Am. Chem. Soc., 120, 12976, 1998 andreferences sited therin.5′-O-DMT-2′-ara-(fluoro)-3′-O-(2-cyanoethyl-N,N-diisopropylamine)-5-methyluridine-phosphoramidite, 5′-O-DMT-2′-ara-(fluoro)-N-6-benzoyl adenosine(3′-O-2-cyanoethyl-N,N-diisopropylamino) phosphoramidite,5′-O-DMT-2′-ara-(fluoro)-N2-isoburytylguanosine-3′-O-(2-cyanoethyl-N,N-diisopropylamino) phosphoramidite and5′-O-DMT-2′-ara-(fluoro)-N-4-benzoylcytidine-3′-O-(2-cyanoethyl-N,N-diisopropylamino) phosphoramidites areobtained by the phosphitylation of the corresponding nucleosides. Thenucleosides are synthesized according to the procedure described byKois,P. et al., Nucleosides Nucleotides 12, 1093, 1993 and Damha et al.,J. Am. Chem. Soc., 120, 12976, 1998. The phosphitylation is carried outby using the bisamidite procedure.

Example 68 Synthesis of 2′-hydroxyl-β-D-arabinofuranosyl compounds

2′-Hydroxyl-D-arabinofuranosyl oligonucleotides are synthesizedfollowing the procedures by Resmini and Pfleiderer Helv. Chim. Acta, 76,158,1993; Schmit et al., Bioorg. Med. Chem. Lett. 4, 1969, 1994 Resmini,M.; Pfleiderer, W. Synthesis of arabinonucleic acid (tANA). Bioorg. Med.Chem. Lett. (1994), 16, 1910.; Resmini, Matthias; Pfleiderer, W.Nucleosides. Part LV. Efficient synthesis of arabinoguanosine buildingblocks (Helv. Chim. Acta, (1994), 77, 429-34; and Damha et al., J. Am.Chem. Soc., 1998, 120, 12976, and references cited therein).

5′-O-DMT-2′-ara-(hydroxy)-3′-O-(2-cyanoethyl-N,N-diisopropylamine)-5-methyluridine-phosphoramidite, 5′-O-DMT-2′-ara-(hydroxy)-N-6-benzoyl adenosine(3′-O-2-cyanoethyl-N,N-diisopropylamino) phosphoramidite,5′-O-DMT-2′-ara-(hydroxy)-N2-isoburytylguanosine-3′-O-(2-cyanoethyl-N,N-diisopropylamino) phosphoramidite and5′-O-DMT-2′-ara-(hydroxy)-N-4-benzoylcytidine-3′-O-(2-cyanoethyl-N,N-diisopropylamino) phosphoramidites areobtained by the phosphitylation of the corresponding nucleosides. Thenucleosides are synthesized according to the procedure described byKois,P. et al., Nucleosides Nucleotides 12, 1093, 1993 and Damha et al.,J. Am. Chem. Soc., 120, 12976, 1998. The phosphitylation is carried outby using the bisamidite procedure.

Example 69 Synthesis of Difluoromethylene Compounds

5′-O-DMT-2′-deoxy-2′-difluoromethylene-5-methyluridine-3′-(2-cyanoethyl-N,N-diisopropylphosphoramidite),5′-O-DMT-2′-deoxy-2′-difluoromethylene-N-4-benzoyl-cytidine,5′-O-DMT-2′-deoxy-2′-diflyoromethylene-N-6-benzoyl adenosine, and5′-O-DMT-2′-deoxy-2′-difluoroethylene-N₂-isobutyryl guanosine aresynthesized following the protocols described by Usman et. al. (U.S.Pat. No. 5,639,649, Jun. 17, 1997).

Example 70 Synthesis of5′-O-DMT-2′-deoxy-2′-β-(O-acetyl)-2′-α-methyl-N6-benzopyl-adenosine-3′-(2-cyanoethyl-N,N-diisopropyl phosphoramidite

5′-O-DMT-2′-deoxy-2′-(OH)-2′-α-methyl-adenosine is synthesized from thecompound 5′-3′-protected-2′-keto-adenosine (Rosenthal, Sprinzl andBaker, Tetrahedron Lett. 4233, 1970; see also Nucleic acid relatedcompounds. A convenient procedure for the synthesis of 2′- and3′-ketonucleosides is shown Hansske et al., Dep. Chem., Univ. Alberta,Edmonton, Can., Tetrahedron Lett. (1983), 24(15), 1589-92.) by Grigandaddition of MeMgI in THF solvent, followed by seperation of the isomers.The 2-(OH) is protected as acetate. 5′-3′-acetal group is removed,5′-position dimethoxy, tritylated, N-6 position is benzoylated and then3′-position is phosphitylated to give5′-O-DMT-2′-deoxy-2′-β-(O-acetyl)-2′-α-methyl-N6-benzoyl-adenosine-3′-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite.

Example 71 Synthesis of5′-O-DMT-2′-α-ethynyl-N6-benzoyl-adenosine-3′-(2-cyanoethyl-N,N-diisopropylphosphoramidite

5′-O-DMT-2′-deoxy-2′-O—(OH)-2′-ethynyl-adenosine is synthesized from thecompound 5′-3′-protected-2′-keto-adenosine (Rosenthal, Sprinzl andBaker, Tetrahedron Lett. 4233, 1970) by Grigand addition of Ethynyl-MgIin THF solvent, followed by seperation of the isomers. The 2′-(OH) isremoved by Robins' deoxygenation procedure (Robins et al., J. Am. Chem.Soc. (1983), 105, 4059-65. 5′-3′-acetal group is removed, 5′-positiondimethoxytritylated, N-6 position is benzoylated and then 3′-position isphosphitylated to give the title compound.

Example 72 2′-O-(guaiacolyl)-5-methyluridine

2-Methoxyphenol (6.2 g, 50 mmol) was slowly added to a solution ofborane in tetrahydrofuran (1 M, 10 mL, 10 mmol) with stirring in a 100mL bomb. Hydrogen gas evolved as the solid dissolvedO-2,2′-anhydro-5-methyluridine (1.2 g, 5 mmol), and sodium bicarbonate(2.5 mg) were added and the bomb was sealed, placed in an oil bath andheated to 155° C. for 36 hours. The bomb was cooled to room temperatureand opened. The crude solution was concentrated and the residuepartitioned between water (200 mL) and hexanes (200 mL). The excessphenol was extracted into hexanes. The aqueous layer was extracted withethyl acetate (3×200 mL) and the combined organic layer was washed oncewith water, dried over anhydrous sodium sulfate and concentrated. Theresidue was purified by silica gel flash column chromatography usingmethanol:methylene chloride ( 1/10, v/v) as the eluent. Fractions werecollected and the target fractions were concentrated to give 490 mg ofpure product as a white solid. Rf=0.545 in CH₂Cl₂/CH₃OH (10:1). MS/ESfor C_(l7)H₂0N₂O₇, 364.4; Observed 364.9.

Example 735′-Dimethoxytrityl-2′-O-(2-methoxyphenyl)-5-methyluridine-3′-O-(2-cyanoethyl-N,N-diisopropylamino)phosphoramidite

2′-O-(guaiacolyl)-5-methyl-uridine is treated with 1.2 equivalents ofdimethoxytrityl chloride (DMT-Cl) in pyridine to yield the5′-O-dimethoxy tritylated nucleoside. After evaporation of the pyridineand work up (CH₂Cl₂/saturated NaHCO₃ solution) the compound is purifiedin a silica gel column. The 5′-protected nucleoside is dissolved inanhydrous methylene chloride and under argon atmosphere,N,N-diisopropylaminohydro-tetrazolide (0.5 equivalents) andbis-N,N-diisopropylamino-2-cyanoethyl-phosphoramidite (2 equivalents)are added via syringe over 1 min. The reaction mixture is stirred underargon at room temperature for 16 hours and then applied to a silicacolumn. Elution with hexane:ethylacetate (25:75) gives the titlecompound.

Example 745′-Dimethoxytrityl-2′-O-(2-methoxyphenyl)-5-methyluridine-3′-O-succinate

The 5′-protected nucleoside from Example 73 is treated with 2equivalents of succinic anhydride and 0.2 equivalents of4-N,N-dimethylaminopyridine in pyridine. After 2 hours the pyridine isevaporated, the residue is dissolved in CH₂Cl₂ and washed three timeswith 100 mL of 10% citric acid solution. The organic layer is dried overanhydrous MgSO₄ to give the desired succinate. The succinate is thenattached to controlled pore glass (CPG) using established procedures(Pon, R. T., Solid phase supports for oligonucleotide synthesis, inProtocols for Oligonucleotides and Analogs, S. Agrawal (Ed.), HumanaPress: Totawa, N.J., 1993, 465-496).

Example 75 5′-Dimethoxytrityl-2′-O-(trans-2-methoxycyclohexyl)-5-methyluridine

2′-3′-O-Dibutylstannyl-5-methyl uridine (Wagner et al., J. Org. Chem.,1974, 39, 24) is alkylated with trans-2-methoxycyclohexyl tosylate at70° C. in DMF. A 1:1 mixture of 2′-O- and3′-O-(trans-2-methoxycyclohexyl)-5-methyluridine is obtained in thisreaction. After evaporation of the DMF solvent, the-crude mixture isdissolved in pyridine and treated with dimethoxytritylchloride (DMT-Cl)(1.5 equivalents). The resultant mixture is purified by silica gel flashcolumn chromatography to give the title compound.

Example 765′-Dimethoxytrityl-2′-O-(trans-2-methoxycyclohexyl)-5-methyluridine-3′-O-(2-cyanoethyl-N,N-diisopropylamino)phosphoramidite

5′-Dimethoxytrityl-2′-O-(trans-2-methoxycyclohexyl)-5-methyl uridine isphosphitylated according to the procedure described above to give therequired phosphoramidite.

Example 775′-Dimethoxytrityl-2′-O-(trans-2-methoxycyclohexyl)-5-methyluridine-3′-O-(succinyl-amino)CPG

5′-Dimethoxytrityl-2′-O-(trans-2-methoxycyclohexyl)-5-methyl uridine issuccinylated and attached to controlled pore glass to give the solidsupport bound nucleoside.

Example 78 Trans-2-ureido-cyclohex

Trans-2-amino-cyclohexanol (Aldrich) is treated with triphosgene inmethylene chloride (⅓ equivalent). To the resulting solution, excessammonium hydroxide is added to give after work up the title compound.

Example 79 2′-O-(trans-2-uriedo-cyclohexyl)-5-methyl uridine

Trans-2-uriedo-cyclohexanol (50 mmol) is added to a solution of boranein tetrahydrofuran (1 M, 10 mL, 10 mmol) while stirring in a 10 mL bomb.Hydrogen gas evolves as the reactant dissolves.O2,2′-Anhydro-5-methyluridine (5 mmol) and sodium bicarbonate (2.5 mg)are added to the bomb and sealed. Then it is heated to 140 for 72 hrs.The bomb is cooled to room temperature and opened. The crude materialwas worked up as illustrated above followed by purification by silicagel flash column chromatography to give the title compound.

Example 80 5′-O-(Dimethoxytrityl)-2′-O-(trans-2-uriedo-cyclohexyl)3′-O-(2-cyanoethyl, N,N,-diisopropyl) uridine phosphoramidite

2′-O-(trans-2-uriedo-cyclohexyl)-5-methyl uridine tritylated at the5′-OH and phosphitylated at the 3′-OH following the proceduresillustrated in example 2 to give the title compound.

Example 815′-O-dimethoxytrityl-2′-O-(trans-2-uriedo-cyclohexyl)-5-methyl-3′-O-(succinyl)-aminoCPG uridine

5′-O-dimethoxytrityl-2′-O-(trans-2-uriedo-cyclohexyl)-5-methyl uridineis succinylated and attached to CPG as illustrated above.

Example 82 2′-O-(trans-2-methoxy-cyclohexyl) adenosine

Trans-2-methoxycyclopentanol, trans-2-methoxycylcohexanol,trans-2-methoxy-cyclopentyl tosylate and trans-2-methoxy-cyclohexyltosylate are prepared according to reported procedures (Roberts, D. D.,Hendrickson, W., J. Org. Chem., 1967, 34, 2415-2417; J. Org. Chem.,1997, 62, 1857-1859). A solution of adenosine (42.74 g, 0.16 mol) in drydimethylformamide (800 mL) at 5° C. is treated with sodium hydride (8.24g, 60% in oil prewashed thrice with hexanes, 0.21 mol). After stirringfor 30 min, trans-2-methoxycyclohexyl tosylate (0.16 mol) is added over20 minutes at 5° C. The reaction is stirred at room temperature for 48hours, then filtered through Celite. The filtrate is concentrated underreduced pressure followed by coevaporation with toluene (2×100 mL) togive the title compound.

Example 83 N⁶-Benzoyl-2′-O-(trans-2-methoxycyclohexyl) adenosine

A solution of 2′-O-(trans-2-methoxy-cyclohexyl) adenosine (0.056 mol) inpyridine (100 mL) is evaporated under reduced pressure to dryness. Theresidue is redissolved in pyridine (560 mL) and cooled in an ice waterbath. Trimethylsilyl chloride (36.4 mL, 0.291 mol) is added and thereaction is stirred at 5° C. for 30 minutes. Benzoyl chloride (33.6 mL,0.291 mol) is added and the reaction is allowed to warm to 25° C. for 2hours and then cooled to 5° C. The reaction is diluted with cold water(112 mL) and after stirring for 15 min, concentrated ammonium hydroxide(112 mL). After 30 min, the reaction is concentrated under reducedpressure (below 30° C.) followed by coevaporation with toluene (2×100mL). The residue is dissolved in ethyl acetate-methanol (400 mL, 9:1)and the undesired silyl by-products are removed by filtration. Thefiltrate is concentrated under reduced pressure and purified by silicagel flash column chromatography (800 g, chloroform-methanol 9:1).Selected fractions are combined, concentrated under reduced pressure anddried at 25° C./0.2 mmHg for 2 h to give the title compound.

Example 84N⁶-Benzoyl-5′-O-(dimethoxytrityl)-2′-O-(trans-2-methoxycyclohexyl)adenosine

A solution of N⁶-benzoyl-2′-O-(trans-2-methoxycyclohexyl) adenosine(0.285 mol) in pyridine (100 mL) is evaporated under reduced pressure toan oil. The residue is redissolved in dry pyridine (300 mL) and4,4′-dimethoxytriphenylmethyl chloride (DMT-Cl, 10.9 g, 95%, 0.31 mol)added. The mixture is stirred at 25° C. for 16 h and then poured onto asolution of sodium bicarbonate (20 g) in ice water (500 mL). The productis extracted with ethyl acetate (2×150 mL). The organic layer is washedwith brine (50 mL), dried over sodium sulfate (powdered) and evaporatedunder reduced pressure (below 40° C.). The residue is chromatographed onsilica gel (400 g, ethyl acetate-hexane 1:1. Selected fractions werecombined, concentrated under reduced pressure and dried at 25° C./0.2mmHg to give the title compound.

Example 85[N⁶-Benzoyl-5′-O-(4,4′-dimethoxytrityl)-2′-O-(trans-2-methoxycyclohexyl)adenosine-3′-O-yl]-N,N-diisopropylamino-cyanoethoxy phosphoramidite

Phosphitylation ofN⁶-benzoyl-5′-O-(dimethoxytrityl)-2′-O-(trans-2-methoxycyclohexyl)adenosine was performed as illustrated above to give the title compound.

Example 86 General procedures for chimeric C3′-endo and C2′-endomodified oligonucleotide synthesis

Oligonucleotides are synthesized on a PerSeptive Biosystems Expedite8901 Nucleic Acid Synthesis System. Multiple 1-mmol syntheses areperformed for each oligonucleotide. The 3′-end nucleoside containingsolid support is loaded into the column. Trityl groups are removed withtrichloroacetic acid (975 mL over one minute) followed by anacetonitrile wash. The oligonucleotide is built using a modified diester(P═O) or thioate (P═S) protocol.

Phosphodiester Protocol

All standard amidites (0.1 M) are coupled over a 1.5 minute time frame,delivering 105 μL material. All novel amidites are dissolved in dryacetonitrile (100 mg of amidite/1 mL acetonitrile) to give approximately0.08-0.1 M solutions. The 2′-modified amidites (both ribo and arabinomonomers) are double coupled using 210 μL over a total of 5 minutes.Total coupling time is approximately 5 minutes (210 mL of amiditedelivered). 1-H-tetrazole in acetonitrile is used as the activatingagent. Excess amidite is washed away with acetonitrile.(1S)-(+)-(10-camphorsulfonyl) oxaziridine (CSO, 1.0 g CSO/8.72 mL dryacetonitrile) is used to oxidize (3 minute wait step) deliveringapproximately 375 μL of oxidizer. Standard amidites are delivered (210μL) over a 3-minute period.

Phosphorothioate Protocol

The 2′-modified amidite is double coupled using 210 μL over a total of 5minutes. The amount of oxidizer, 3H-1,2-benzodithiole-3-one-1,1-dioxide(Beaucage reagent, 3.4 g Beaucage reagent/200 mL acetonitrile), is 225μL (one minute wait step). The unreacted nucleoside is capped with a50:50 mixture of tetrahydrofuran/acetic anhydride andtetrahydrofuran/pyridine/1-methyl imidazole. Trityl yields are followedby the trityl monitor during the duration of the synthesis. The finalDMT group is left intact. After the synthesis, the contents of thesynthesis cartridge (1 mmole) is transferred to a Pyrex vial and theoligonucleotide is cleaved from the controlled pore glass (CPG) using30% ammonium hydroxide (NH₄OH, 5 mL) for approximately 16 hours at 55°C.

Oligonucleotide Purification

After the deprotection step, the samples are filtered from CPG usingGelman 0.45 μm nylon acrodisc syringe filters. Excess NH₄OH isevaporated away in a Savant AS160 automatic speed vac. The crude yieldis measured on a Hewlett Packard 8452A Diode Array Spectrophotometer at260 nm. Crude samples are then analyzed by mass spectrometry (MS) on aHewlett Packard electrospray mass spectrometer. Trityl-onoligonucleotides are purified by reverse phase preparative highperformance liquid chromatography (HPLC). HPLC conditions are asfollows: Waters 600E with 991 detector; Waters Delta Pak C4 column(7.8×300 mm); Solvent A: 50 mM triethylammonium acetate (TEA-Ac), pH7.0; Solvent B: 100% acetonitrile; 2.5 mL/min flow rate; Gradient: 5% Bfor first five minutes with linear increase in B to 60% during the next55 minutes. Fractions containing the desired product/s (retentiontime=41 minutes for DMT-ON-16314; retention time=42.5 minutes forDMT-ON-16315) are collected and the solvent is dried off in the speedvac. Oligonucleotides are detritylated in 80% acetic acid forapproximately 60 minutes and lyophilized again. Free trityl and excesssalt are removed by passing detritylated oligonucleotides throughSephadex G-25 (size exclusion chromatography) and collecting appropriatesamples through a Pharmacia fraction collector. The solvent is againevaporated away in a speed vac. Purified oligonucleotides are thenanalyzed for purity by CGE, HPLC (flow rate: 1.5 mL/min; Waters DeltaPak C4 column, 3.9×300 mm), and MS. The final yield is determined byspectrophotometer at 260 nm.

Example 87 Rapid amplification of 5′-cDNA end (5′-RACE) and 3′-cDNA end(3′-RACE)

An internet search of the XREF database in the National Center ofBiotechnology Information (NCBI) yielded a 361 base pair (bp) humanexpressed sequenced tag (EST, GenBank accession #H28861), homologous toyeast RNase H (RNH1) protein sequenced tag (EST, GenBank accession#Q04740) and its chicken homologue (accession #D26340). Three sets ofoligonucleotide primers encoding the human RNase H EST sequence weresynthesized. The sense primers were ACGCTGGCCGGGAGTCGAAATGCTTC (H1: SEQID NO: 6), CTGTTCCTGGCCCACAGAGTCGCCTTGG (H3: SEQ ID NO: 7) andGGTCTTTCTGACCTGGAATGAGTGCAGAG (H5: SEQ ID NO: 8). The antisense primerswere CTTGCCTGGTTTCGCCCTCCGATTCTTGT (H2: SEQ ID NO: 9),TTGATTTTCATGCCCTTCTGAAACTTCCG (H4; SEQ ID NO: 10) andCCTCATCCTCTATGGCAAACTTCTTAAATCTGGC (H6; SEQ ID NO: 11). The human RNaseH 3′ and 5′ cDNAs derived from the EST sequence were amplified bypolymerase chain reaction (PCR), using human liver or leukemia(lymphoblastic Molt-4) cell line Marathon ready cDNA as templates, H1 orH3/AP1 as well as H4 or H6/AP2 as primers (Clontech, Palo Alto, Calif.).The fragments were subjected to agarose gel electrophoresis andtransferred to nitrocellulose membrane (Bio-Rad, Hercules Calif.) forconfirmation by Southern blot, using ³²P-labeled H2 and H1 probes (for3′ and 5′ RACE products, respectively, in accordance with proceduresdescribed by Ausubel et al., Current Protocols in Molecular Biology,Wiley and Sons, New York, N.Y., 1988. The confirmed fragments wereexcised from the agarose gel and purified by gel extraction (Qiagen,Germany), then subcloned into Zero-blunt vector (Invitrogen, Carlsbad,Calif.) and subjected to DNA sequencing.

Example 88 Screening of the cDNA Library, DNA Sequencing and SequenceAnalysis

A human liver cDNA lambda phage Uni-ZAP library (Stratagene, La Jolla,Calif.) was screened using the RACE products as specific probes. Thepositive cDNA clones were excised into the pBluescript phagemid(Stratagene, La Jolla Calif.) from lambda phage and subjected to DNAsequencing with an automatic DNA sequencer (Applied Biosystems, FosterCity, Calif.) by Retrogen Inc. (San Diego, Calif.). The overlappingsequences were aligned and combined by the assembling programs ofMacDNASIS v3.0 (Hitachi Software Engineering America, South SanFrancisco, Calif.). Protein structure and subsequence analysis wereperformed by the program of MacVector 6.0 (Oxford Molecular Group Inc.,Campbell, Calif.). A homology search was performed on the NCBI databaseby internet E-mail.

Example 89 Northern Blot and Southern Blot Analysis

Total RNA from different human cell lines (ATCC, Rockville, Md.) wasprepared and subjected to formaldehyde agarose gel electrophoresis inaccordance with procedures described by Ausubel et al., CurrentProtocols in Molecular Biology, Wiley and Sons, New York, N.Y., 1988,and transferred to nitrocellulose membrane (Bio-Rad, Hercules Calif.).Northern blot hybridization was carried out in QuickHyb buffer(Stratagene, La Jolla, Calif.) with ³²P-labeled probe of full lengthRNase H cDNA clone or primer H1/H2 PCR-generated 322-base N-terminalRNase H cDNA fragment at 68° C. for 2 hours. The membranes were washedtwice with 0.1% SSC/0.1% SDS for 30 minutes and subjected toauto-radiography. Southern blot analysis was carried out in 1×pre-hybridization/hybridization buffer (BRL, Gaithersburg, Md.) with a³²P-labeled 430 bp C-terminal restriction enzyme PstI/PvuII fragment or1.7 kb full length cDNA probe at 60° C. for 18 hours. The membranes werewashed twice with 0.1% SSC/0.1% SDS at 60° C. for 30 minutes, andsubjected to autoradiography.

Example 90 Expression and Purification of the Cloned RNase Protein

The cDNA fragment coding the full RNase H protein sequence was amplifiedby PCR using 2 primers, one of which contains restriction enzyme NdeIsite adapter and six histidine (his-tag) codons and 22 bp protein Nterminal coding sequence. The fragment was cloned into expression vectorpETI 7b (Novagen, Madison, Wis.) and confirmed by DNA sequencing. Theplasmid was transfected into E.coli BL21 (DE3) (Novagen, Madison, Wis.).The bacteria were grown in M9ZB medium at 32EC and harvested when theOD₆₀₀ of the culture reached 0.8, in accordance with proceduresdescribed by Ausubel et al., Current Protocols in Molecular Biology,Wiley and Sons, New York, N.Y., 1988. Cells were lysed in 8M ureasolution and recombinant protein was partially purified with Ni-NTAagarose (Qiagen, Germany). Further purification was performed with C4reverse phase chromatography (Beckman, System Gold, Fullerton, Calif.)with 0.1% TFA water and 0.1% TFA acetonitrile gradient of 0% to 80% in40 minutes as described by Deutscher, M. P., Guide to ProteinPurification, Methods in Enzymology 182, Academic Press, New York, N.Y.,1990. The recombinant proteins and control samples were collected,lyophilized and subjected to 12% SDS-PAGE as described by Ausubel et al.(1988) Current Protocols in Molecular Biology, Wiley and Sons, New York,N.Y. The purified protein and control samples were resuspended in 6 Murea solution containing 20 mM Tris HCl, pH 7.4, 400 mM NaCl, 20%glycerol, 0.2 mM PMSF, 5 mM DTT, 10 μg/ml aprotinin and leupeptin, andrefolded by dialysis with decreasing urea concentration from 6 M to 0.5M as well as DTT concentration from 5 mM to 0.5 mM as described byDeutscher, M. P., Guide to Protein Purification, Methods in Enzymology182, Academic Press, New York, N.Y., 1990. The refolded proteins wereconcentrated (10 fold) by Centricon (Amicon, Danvers, Mass.) andsubjected to RNase H activity assay.

Example 91 RNase H Activity Assay

³²P-end-labeled 17-mer RNA, GGGCGCCGTCGGTGTGG (SEQ ID NO: 12) describedby Lima, W. F. and Crooke, S. T., Biochemistry, 1997 36, 390-398, wasgel-purified as described by Ausubel et al., Current Protocols inMolecular Biology, Wiley and Sons, New York, N.Y., 1988 and annealedwith a tenfold excess of its complementary 17-mer oligodeoxynucleotideor a 5-base DNA gapmer, i.e., a 17 mer oligonucleotide which has acentral portion of 5 deoxynucleotides (the “gap”) flanked on both sidesby 6 2′-methoxynucleotides. Annealing was done in 10 mM Tris HCl, pH8.0, 10 mM MgCl, 50 mM KCl and 0.1 mM DTT to form one of three differentsubstrates: (a) single strand (ss) RNA probe, (b) full RNA/DNA duplexand (c) RNA/DNA gapmer duplex. Each of these substrates was incubatedwith protein samples at 37° C. for 5 minutes to 2 hours at the sameconditions used in the annealing procedure and the reactions wereterminated by adding EDTA in accordance with procedures described byLima, W. F. and Crooke, S. T., Biochemistry, 1997, 36, 390-398. Thereaction mixtures were precipitated with TCA centrifugation and thesupernatant was measured by liquid scintillation counting (BeckmanLS6000IC, Fullerton, Calif.). An aliquot of the reaction mixture wasalso subjected to denaturing (8 M urea) acrylamide gel electrophoresisin accordance with procedures described by Lima, W. F. and Crooke, S.T., Biochemistry, 1997, 36, 390-398 and Ausubel et al., CurrentProtocols in Molecular Biology, Wiley and Sons, New York, N.Y., 1988.

Example 92 Effects of Phosphorothioate Substitution and Substrate Lengthon Digestion by Human RNase H1 (See Table 4)

Oligoribonucleotides were preannealed with the complementary antisenseoligodeoxynucleotide at 10 nM and 20 nM respectively and subjected todigestion by Human RNase H1. The 17 mer (RNA no.1) and 25 mer (RNA no.3)RNA sequences are derived from Harvy-RAS oncogen 51) and the 25 mer RNAcontains the 17 mer sequence. The 20 mer (RNA no.2) sequence is derivedfrom human hepatitis C virus core protein coding sequence (52). Theinitial rates were determined as described in Materials and Methods, 1A:Comparison of the initial rates of cleavage of an RNA:phosphodiester(P═O) and an RNA:phosphorothioate (P═S) duplexes, and 1B: Comparisonamong duplexes of different sequences and lengths.

Example 93 Effects of 2′-Substitution and Deoxy-Gap Size on DigestionRates by Human RNase H1 (See Table 5)

Substrate duplexes were hybridized and initial rates were determined asshown in Table 4 and described in Material and Methods. The 17 mer RNAis the same used in Table 4, and the 20 mer RNA (UGGUGGGCAAUGGGCGUGUU,RNA no.4) is derived from the protein kinase C-zeta (53) sequence. The17 mer and 20 mer P═S oligonucleotides were full deoxyphosphorothioatecontaining no 2′-modifications. The 9, 7, 5, 4 and 3 deoxy gapoligonucleotides were 17 mer oligonucleotide with a central portionconsisting of nine, seven, five and four deoxynucleotides flanked onboth sides by 2′-methoexynucleotides (also see FIG. 2). Boxed sequencesindicate the position of the 2′-methoxy-modified residues. Dash-boxedsequence indicates the position of the 2′-propoxy-modified residues.

Example 94 Kinetic Constants for RNase H1 Cleavage of RNA:DNA Duplexes(See Table 6)

The RNA:DNA duplexes in Table 4 were used to determine Km and Vmax ofHuman and E.coli RNase H1 as described in the Materials and Methodssection.

Example 95 Binding Constants and Specificity of RNase H's (See Table 7)

K_(d)'s were determined as described in Materials and methods. TheK_(d)'s for E. coli RNase H1 was derived from previously reported data(21). The competing substrates (competitive inhibitors) used in thebinding study are divided into two categories: single-strand (ss)oligonucleotides and oligonucleotide duplexes all with the 17 mersequence as in Table 4 (RNA No. 1). The single-strand oligonucleotidesincluded: ssRNA, ssDNA, ss fully modified 2′-methoxy phosphodiesteroligonucleotide (ss 2′-O-Me) and ss full phosphorothioatedeoxyoligonucleotide (ss DNA, P═S). The duplex substrates include:DNA:DNA duplex, RNA:RNA duplex, DNA:fully modified 2′ fluoro or fullymodified 2′-methoxy oligonucleotide (DNA: 2′-F or 2′-O-Me), RNA: 2′-F or2′-O-Me duplex. Dissociation constants are derived from 3 slopes ofLineweaver-Burk and /or Augustisson analysis. Estimated errors for thedissociation constants are 2 fold. Specificity is defined by dividingthe K_(d) for a duplex by the K_(d) for an RNA:RNA duplex. TABLE 4 A RNAAnitsense Initial Rate 1 GGGCGCCGUCGGUGU 17 mer 1050 ± 203 GG P═O 1GGGCGCCGUCGGUGU 17 mer 4034 ± 266 GG P═S B Initial Rate RNA Antisense(pmol No. RNA DNA L⁻¹ min⁻¹) 1 GGGCGCCGUCGGUGUGG 17 mer 1050 ± 203 (SEQID NO: 21) P═O 2 ACUCCACCAUAGUACACUCC 20 mer 1015 ± 264 (SEQ ID NO: 22)P═O 3 UGGUGGGCGCCGUCGGUGUGGCAA 25 mer 1502 ± 182 (SEQ ID NO: 23) P═O

TABLE 5 Initial Rate RNA (pmol No. RNA Antisense DNA L⁻¹ min⁻¹) 1 17 merCCACACCGACGGCGCCC 4034 ± 266 (SEQ ID NO: 24) 17 mer CCACACCGACGGCGCCC1081 ± 168 17 mer CCACACCGACGGCGCCC 605 ± 81 17 mer CCACACCGACGGCGCCC330 ± 56 17 mer CCACACCGACGGCGCCC 0 17 mer CCACACCGACGGCGCCC 0 17 merCCACACCGACGGCGCCC 0 4* 20 mer AACACGCCCATTGCCCACCA 3400 ± 384 (SEQ IDNO: 25) 20 mer AACACGCCCATTGCCCACCA 0*Table legend for sequence

TABLE 6 E. coli Human RNase H1 RNase H Vmax Km (nmol Km (nmol Substrates(nM) L⁻¹ min⁻¹) (nM) L⁻¹ min⁻¹) 25 mer Ras 35.4 1.907 (RNA no.3): DNA(P═O) 17 mer Ras 56.1 1.961 385 38.8 (RNA no.1): DNA (P═O) 17 mer Ras13.9 1.077 (RNA no.1): DNA (P═S)

TABLE 7 Human RNase H E. Coli RNase H1 Inhibitors Kd(nM) SpecificityKd(nM) Specificity DNA: 2′-O-Me 458 5.8 3400 2.1 RNA: 2′-O-Me 409 5.23100 1.9 RNA: RNA 79 1.0 1600 1.0 RNA: 2′-F 76 1.0 DNA: 2′-F 99 1.3 DNA:DNA 3608 45.7 176000 110.0 ssRNA 1400 17.7 ssDNA 1506 19.6 942000 588.8ss2′-O-Me 2304 29.2 118000 73.8 ssDNA, P═S 36 0.5 14000 8.8ProceduresProcedure 1ICAM-1 Expression

Oligonucleotide Treatment of HUVECs

Cells were washed three times with Opti-MEM (Life Technologies, Inc.)prewarmed to 37° C. Oligonucleotides were premixed with 10 g/mLLipofectin (Life Technologies, Inc.) in Opti-MEM, serially diluted tothe desired concentrations, and applied to washed cells. Basal anduntreated (no oligonucleotide) control cells were also treated withLipofectin. Cells were incubated for 4 h at 37° C., at which time themedium was removed and replaced with standard growth medium with orwithout 5 mg/mL TNF-7 & D Systems). Incubation at 37° C. was continueduntil the indicated times.

Quantitation of ICAM-1 Protein Expression by Fluorescence-activated CellSorter

Cells were removed from plate surfaces by brief trypsinization with0.25% trypsin in PBS. Trypsin activity was quenched with a solution of2% bovine serum albumin and 0.2% sodium azide in PBS (+Mg/Ca). Cellswere pelleted by centrifugation (1000 rpm, Beckman GPR centrifuge),resuspended in PBS, and stained with 3 1/10⁵ cells of the ICAM-1specific antibody, CD54-PE (Pharmingin). Antibodies were incubated withthe cells for 30 min at 4C in the dark, under gently agitation. Cellswere washed by centrifugation procedures and then resuspended in 0.3 mLof FacsFlow buffer (Becton Dickinson) with 0.5% formaldehyde(Polysciences). Expression of cell surface ICAM-1 was then determined byflow cytometry using a Becton Dickinson FACScan. Percentage of thecontrol ICAM-1 expression was calculated as follows:[(oligonucleotide-treated ICAM-1 value)—(basal ICAM-1value)/(non-treated ICAM-1 value)—(basal ICAM-1 value)]. (Baker, Brenda,et. al. 2′-O-(2-Methoxy)ethyl-modified Anti-intercellular AdhesionMolecule 1 (ICAM-1) Oligonucleotides Selectively Increase the ICAM-1mRNA Level and Inhibit Formation of the ICAM-1 Translation InitiationComplex in Human Umbilical Vein Endothelial Cells, The Journal ofBiological Chemistry, 272, 11994-12000, 1997.)

ICAM-1 expression of chimeric C3′-endo and C2′-endo modifiedoligonucleotides of the invention is measured by the reduction of ICAM-1levels in treated HUVEC cells. The oligonucleotides are believed to workby RNase H cleavage mechanism. Appropriate scrambled controloligonucleotides are used as controls. They have the same basecomposition as the test sequence.

Sequences that contain the chimeric C3′-endo (2′-MOE)and C2′-endo (oneof the following modifications: 2′-S-Me, 2′-Me, 2′-ara-F,2′-ara-OH,2′-ara-O-Me) as listed in Table X below are prepared and tested in theabove assay. SEQ ID NO: 43, a C-raf targeted oligonucleotide, is used asa control. TABLE X Oligonucleotides Containing chimeric2′-O-(2-methoxyethyl) and 2′-S-(methyl) modifications. SEQ ID NO:Sequence (5′-3′) Target 43 AsTsGs C ^(m) sAsTs TsCs^(m)Ts GsCs_(m) mouseCs^(m) Cs^(m)C^(m)sC ^(m) s AsAsGs GsA C-raf 44 GsC^(m)sC^(m)sC^(m)sAsAs GsC^(m)sTs Human GsGsC^(m)s ASTsC^(m)S C^(m)sGSTs ICAM-1C^(m)SAAll nucleosides in bold are 2′-O-(methoxyethyl); subscript s indicates aphosphorothioate linkage; underlined nucleosides indicate 2′-S-Me-modification. superscript m on C (Cm)indicates a 5-methyl-C.

TABLE XI Oligonucleotides Containing chimeric 2′-O-(2-methoxyethyl) and2′-O-(methyl) modifications SEQ ID NO: Sequence (5′-3′) Target 43 AsTsGsC^(m)sAsTs TsCs^(m)Ts Mouse GsCs^(m)Cs^(m) Cs^(m)C^(m)sC^(m)s AsAsGsC-raf GsA 44 GsC^(m)sC^(m)s C^(m)sAsAs GsC^(m)sTs Human GsGsC^(m)sASTsC^(m)S C^(m)sGSTs ICAM-1 C^(m)SAAll nucleosides in bold are 2′-O-(methoxyethyl); subscript s indicates aphosphorothioate linkage; underlined nucleosides indicate 2′-Methylmodification. Superscript m on C (Cm)indicates a 5-methyl-C.

TABLE XII Oligonucleotides Containing chimeric 2′-O-(2-methoxyethyl) and2′-ara-(fluoro) modifications SEQ ID NO: Sequence (5′-3′) Target 43AsTsGs C^(m)sAsTs TsCs^(m)Ts mouse GsCs^(m)Cs^(m) Cs^(m)C^(m)sC^(m)sAsAsGs C-raf GsA 44 GsC^(m)sC^(m)s C^(m)sAsAs GsC^(m)sTs humanGsGsC^(m)s ASTsC^(m)S C^(m)sGSTs ICAM-1 C^(m)SAAll nucleosides in bold are 2′-O-(methoxyethyl); subscript s indicates aphosphorothioate linkage; underlined nucleosides indicate2′-ara-(fluoro) modification. superscript m on C (Cm)indicates a5-methyl-C.

TABLE XIII Oligonucleotides Containing chimeric 2′-O-(2-methoxyethyl)and 2′-ara-(OH) modifications SEQ ID NO: Sequence (5′-3′) Target 43AsTsGs C^(m)sAsTs TsCs^(m)Ts mouse GsCs^(m)Cs^(m) Cs^(m)C^(m)sC^(m)sAsAsGs C-raf GsA 44 GsC^(m)sC^(m)s C^(m)sAsAs GsC^(m)sTs humanGsGsC^(m)s ASTsC^(m)S C^(m)sGSTs ICAM-1 C^(m)SAAll nucleosides in bold are 2=-O-(methoxyethyl); subscript s indicates aphosphorothioate linkage; underlined nucleosides indicate 2′-ara-(OH)modification. superscript m on C (Cm)indicates a 5-methyl-C.

TABLE XIV Oligonucleotides Containing chimeric 2′-O-(2-methoxyethyl) and2′-ara-(OMe) modifications SEQ ID NO: Sequence (5′-3′) Target 43 AsTsGsC^(m)sAsTs TsCs^(m)Ts mouse GsCs^(m)Cs^(m) Cs^(m)C^(m)sC^(m)s AsAsGsC-raf GsA 44 GsC^(m)sC^(m)s C^(m)sAsAs GsC^(m)sTs human GsGsC^(m)sASTsC^(m)S C^(m)sGSTs ICAM-1 C^(m)SAAll nucleosides in bold are 2=-O-(methoxyethyl); subscript S indicates aphosphorothioate linkage; underlined nucleosides indicate 2′-ara-(OMe)modification. superscript m on C (C^(m))indicates a 5-methyl-C.Procedure 2Enzymatic Degradation of 2′-O-Modified Oligonucleotides

Three oligonucleotides are synthesized incorporating the modificationsshown in Table 2 below at the 3′-end. These modified oligonucleotidesare subjected to snake venom phosphodiesterase action.

Oligonucleotides (30 nanomoles) are dissolved in 20 mL-of buffercontaining 50 mM Tris-HCl pH 8.5, 14 mM MgCl₂, and 72 mM NaCl. To thissolution 0.1 units of snake-venom phosphodiesterase (Pharmacia,Piscataway, N.J.), 23 units of nuclease P1 (Gibco LBRL, Gaithersberg,Md.), and 24 units of calf intestinal phosphatase (Boehringer Mannheim,Indianapolis, Ind.) are added and the reaction mixture is incubated at37C for 100 hours. HPLC analysis is carried out using a Waters model 715automatic injector, model 600E pump, model 991 detector, and an Alltech(Alltech Associates, Inc., Deerfield, Ill.) nucleoside/nucleotide column(4.6×250 mm). All analyses are performed at room temperature. Thesolvents used are A: water and B: acetonitrile. Analysis of thenucleoside composition is accomplished with the following gradient: 0-5min., 2% B (isocratic); 5-20 min., 2% B to 10% B (linear); 20-40 min.,10% B to 50% B. The integrated area per nanomole is determined usingnucleoside standards. Relative nucleoside ratios are calculated byconverting integrated areas to molar values and comparing all values tothymidine, which is set at its expected value for each oligomer. TABLEXV Relative Nuclease Resistance of 2′-Modified Chimeric Oligonucleotides5′-TTT TTT TTT TTT TTT T*T*T*T*-3′ SEQ ID NO 45 (Uniform phosphodiester)T* = 2′-modified T-S-Me-Me-2′-ara-(F)-2′-ara-(OH)-2′-ara-(OMe)Procedure 3General Procedure for the Evaluation of Chimeric C3′-Endo and C2′-EndoModified Oligonucleotides Targeted to Ha-ras

Different types of human tumors, including sarcomas, neuroblastomas,leukemias and lymphomas, contain active oncogenes of the ras genefamily. Ha-ras is a family of small molecular weight GTPases whosefunction is to regulate cellular proliferation and differentiation bytransmitting signals resulting in constitutive activation of ras areassociated with a high percentage of diverse human cancers. Thus, rasrepresents an attractive target for anticancer therapeutic strategies.

SEQ ID NO: 46 is a 20-base phosphorothioate oligodeoxynucleotidetargeting the initiation of translation region of human Ha-ras and it isa potent isotype-specific inhibitor of Ha-ras in cell culture based onscreening assays (IC₅₀=45 nm). Treatment of cells in vitro with SEQ IDNO: 46 results in a rapid reduction of Ha-ras mRNA and protein synthesisand inhibition of proliferation of cells containing an activating Ha-rasmutation. When administered at doses of 25 mg/kg or lower by dailyintraperitoneal injection (IP), SEQ ID NO: 46 exhibits potent antitumoractivity in a variety of tumor xenograft models, whereas mismatchcontrols do not display antitumor activity. SEQ ID NO: 46 has been shownto be active against a variety of tumor types, including lung, breast,bladder, and pancreas in mouse xenograft studies (Cowsert, L. M.Anti-cancer drug design, 1997, 12, 359-371). A second-generation analogof SEQ ID NO: 46, where the 5′ and 3′ termini (“wings”) of the sequenceare modified with 2′-methoxyethyl (MOE) modification and the backbone iskept as phosphorothioate (Table XV, SEQ ID NO: 52), exhibits IC₅₀ of 15nm in cell culture assays. thus, a 3-fold improvement in efficacy isobserved from this chimeric analog. Because of the improved nucleaseresistance of the 2′-MOE phosphorothioate, SEQ ID NO: 52 increases theduration of antisense effect in vitro. This will relate to frequency ofadministration of this drug to cancer patients. SEQ ID NO: 52 iscurrently under evaluation in ras dependent tumor models (Cowsert, L. M.Anti-cancer drug design, 1997, 12, 359-371). The parent compound, SEQ IDNO: 46, is in Phase I clinical trials against solid tumors by systemicinfusion.

Antisense oligonucleotides having the 2′-Me modification are preparedand tested in the aforementioned assays in the manner described todetermine activity.

Ha-ras Antisense Oligonucleotides With chimeric C3′-endo and C2′-endomodifications and Their Controls. TABLE XV Ha-ras AntisenseOligonucleotides With chimeric C3′-endo and C2′-endo modifications andTheir Controls. Back- 2′- SEQ Sequence ID NO: bone Modif. Comments 465′-TsCsCs GsTsCs P═S 2′-H parent AsTsCs GsCsTs CsCsTs CsAsGs GsG-3′ 475′-TsCsAs GsTsAs AsTsAs P═S 2′-H mismatch GsGsCs CsCsAs CsAsTs controlGsG-3′ 48 5′-ToToCo GsTsCs AsTsCs P═O/ 2′-O-Moe Parent GsCsTs CoCoToCoAoGo P═S/ in wings Gapmer GoG-3′ P═O (Mixed back- bone) 49 5′-TsCsCsGsTsCs AsTsCs P═S 2′-O-MOE Parent GsCsTs CsCsTs CsAsGs in wings GapmerGsG-3′ as Uniform thioate 50 5′-ToCoAo GsTsAs AsTsAs P═O/ 2′-O-MOEParent GsCsCs GsCsCs GsCoCo P═S/ in wings Gapmer CoCoAo CoAoTo GoG-3′P═O (mixed Back- bone) 51 5′-TsCsAs GsTsAs AsTs P═S 2′-O-MOE ControlAs GsCsCs GsCsCs in wings Gapmer CsCsAs CsAsTs GsC-3′ as Uniform Thioate52 5′-TsCsCs GsTsCs AsTsCs P═S 2′-O-MOE Control GsCsTs CsCsTs CsAsGs inwings Gapmer GsG-3′ With MOE Control 53 5′-TsCsAs GsTsAs AsTsAs P═S2′-O-MOE Control GsCsCs GsCsCs CsCsAs in wings Gapmer CsAsTs GsC-3′ WithMOE ControlAll underlined portions of sequences are 2′-Me.Procedure 7In vivo Nuclease Resistance

The in vivo Nuclease Resistance of chimeric C3′-endo and C2′-endomodified oligonucleotides is studied in mouse plasma and tissues (kidneyand liver). For this purpose, the C-raf oligonucleotide series SEQ IDNO: 54 are used and the following five oligonucleotides listed in theTable below will be evaluated for their relative nuclease resistance.TABLE XVI Study of in vivo Nuclease Resistance of chimeric C3′-endo(2′-O-MOE) and C2′-endo (2′-S-Me) modified oligonucleotides with andwithout nuclease resistant caps (2′-5′-phosphate or phosphorothioatelinkage with 3′-O-MOE in cap ends). SEQ ID Back- NO: Sequence boneDescription 54 5′-ATG CAT TCT GCC P═S, (control) CCA AGGA-3′ 2′-H rodentC-raf antisense oligo 55 AoToGo CoAsTs TsCsTs P═O/ 2′-MOE/2′-S-Me/GsCsCs CsCsAo AoGoGo P═S/ 2′-MOE A P═O 56 AsTsGs CsAsTs TsCsTs P═S2′-MOE/2′-S-Me/ GsCsCs CsCsAs AsGsGs 2′-MOE A 57 Ao*ToGo CoAsTs TsCsTsP═O/ In asterisk, GsCsCs CsCsAo AoGoGo P═S/ 2′-5′ linkage with *A P═O3′-O-MOE; 2′-O-MOE/ 2′-S-Me/2′-O-MOE/ 2′-5′ linkage with 3′-O-MOE inasterisk 58 As*TsGs CsAsTs TsCsTs P═S In asterisk, GsCsCs CsCsAs AsGsGs2′-5′ linkage with *A 3′-O-MOE; 2′-O-MOE/ 2′-S-Me/2′-O-MOE/2′-5′ linkage with 3′-O-MOE in asterisk

TABLE XVII Study of in vivo Nuclease Resistance of chimeric C3′-endo(2′-O-MOE) and C2′-endo (2′-Me) modified oligonucleotides with andwithout nuclease resistant caps (2′-5′-phosphate or phosphoro- thioatelinkage with 3′-O-MOE in cap ends). SEQ ID Back- NO: Sequence boneDescription 54 5′-ATG CAT TCT GCC P═S, (control) CCA AGGA-3′ 2′-H rodentC-raf antisense oligo 55 AoToGo CoAsTs TsCsTs P═O/ 2′-MOE/2′-Me/ GsCsCsCsCsAo AoGoGo P═S/ 2′-MOE A′ P═O 56 AsTsGs CsAsTs TsCsTs P═S2′-MOE/2′-Me/ GsCsCs CsCsAs AsGsGs 2′-MOE A 57 Ao*ToGo CoAsTs P═O/ Inasterisk, TsCsTs GsCsCs CsCsAo P═S/ 2′-5′ linkage with AoGoGo *A P═O3′-O-MOE; 2′-O-MOE/ 2′-Me/2′-O-MOE/ 2′-5′ linkage with 3′-O-MOE inasterisk 58 As*TsGs CsAsTs P═S In asterisk, TsCsTs GsCsCs CsCsAs2′-5′ linkage with AsGsGs *A 3′-O-MOE; 2′-O-MOE/ 2′-Me/2′-O-MOE/2′-5′ linkage with 3′-O-MOE in asterisk

TABLE XVIII Study of in vivo Nuclease Resistance of chimeric C3′-endo(2′-O-MOE) and C2′-endo (2′-ara-F) modified oligonucleotides with andwithout nuclease resistant caps (2′-5′-phosphate or phosphorothioatelinkage with 3′-O-MOE in cap ends). SEQ ID Back- NO: Sequence boneDescription 54 5′-ATG CAT TCT GCC P═S, (control) CCA AGGA-3′ 2′-H rodentC-raf antisense oligo 55 AoToGo CoAsTs TsCsTs P═O/ 2′-MOE/2′-ara-F/GsCsCs CsCsAo AoGoGo P═S/ 2′-MOE A′ P═O 56 AsTsGs CsAsTs TsCsTs P═S2′-MOE/2′-ara-F/ GsCsCs CsCsAs AsGsGs 2′-MOE A 57 Ao*ToGo CoAsTs TsCsTsP═O/ In asterisk, GsCsCs CsCsAo AoGoGo P═S/ 2′-5′ linkage with *A P═O3′-O-MOE; 2′-O-MOE/ 2′-ara-F/2′-O-MOE/ 2′-5′ linkage with 3′-O-MOE inasterisk 58 As*TsGs CsAsTs TsCsTs P═S In asterisk, GsCsCs CsCsAs AsGsGs2′-5′ linkage with *A 3′-O-MOE; 2′-O-MOE/ 2′-ara-F/2′-O-MOE/2′-5′ linkage with 3′-O-MOE in asterisk

TABLE XIX Study of in vivo Nuclease Resistance of chimeric C3′-endo(2′-O-MOE) and C2′-endo (2′-ara-OH) modified oligonucleotides with andwithout nuclease resistant caps (2′-5′-phosphate or phosphorothioatelinkage with 3′-O-MOE in cap ends). SEQ ID Back- NO: Sequence boneDescription 54 5′-ATG CAT TCT GCC P═S, (control) CCA AGGA-3′ 2′-H rodentC-raf antisense oligo 55 AoToGo CoAsTs TsCsTs P═O/ 2′-MOE/2′-ara-OH/GsCsCs CsCsAo AoGoGo P═S/ 2′-MOE A′ P═O 56 AsTsGs CsAsTs TsCsTs P═S2′-MOE/2′-ara-OH/ GsCsCs CsCsAs AsGsGs 2′-MOE A 57 Ao*ToGo CoAsTs TsCsTsP═O/ In asterisk, GsCsCs CsCsAo AoGoGo P═S/ 2′-5′ linkage with *A P═O3′-O-MOE; 2′-O-MOE/ 2′-ara-OH/2′-O-MOE/ 2′-5′ linkage with 3′-O-MOE inasterisk 58 As*TsGs CsAsTs TsCsTs P═S In asterisk, GsCsCs CsCsAs AsGsGs2′-5′ linkage with *A 3′-O-MOE; 2′-O-MOE/ 2′-ara-OH/2′-O-MOE/2′-5′ linkage with 3′-O-MOE in asterisk

TABLE XX Study of in vivo Nuclease Resistance of chimeric C3′-endo(2′-O-MOE) and C2′-endo (2′-ara-OMe) modified oligonucleotides with andwithout nuclease resistant caps (2′-5′-phosphate or phosphorothioatelinkage with 3′-O-MOE in cap ends). SEQ ID Back- NO: Sequence boneDescription 54 5′-ATG CAT TCT GCC P═S, (control) CCA AGGA-3′ 2′-H rodentC-raf antisense oligo 55 AoToGo CoAsTs TsCsTs P═O/ 2′-MOE/2′-ara-OMe/GsCsCs CsCsAo AoGoGo P═S/ 2′-MOE A′ P═O 56 AsTsGs CsAsTs TsCsTs P═S2′-MOE/2′-ara-OMe/ GsCsCs CsCsAs AsGsGs 2′-MOE A 57 Ao*ToGo CoAsTsTsCsTs P═O/ In asterisk, GsCsCs CsCsAo AoGoGo P═S/ 2′-5′ linkage with *AP═O 3′-O-MOE; 2′-O-MOE/ 2′-ara-OMe/2′-O- MOE/2′-5′ linkage with 3′-O-MOEin asterisk 58 As*TsGs CsAsTs TsCsTs P═S In asterisk, GsCsCs CsCsAsAsGsGs 2′-5′ linkage with *A 3′-O-MOE; 2′-O-MOE/ 2′-ara-OMe/2′-O-MOE/2′-5′ linkage with 3′-O-MOE in asteriskProcedure 8Animal Studies for in vivo Nuclease Resistance

For each oligonucleotide to be studied, 9 male BALB/c mice (CharlesRiver, Wilmington, Mass.), weighing about 25 g are used (Crooke et al.,J. Pharmacol. Exp. Ther., 1996, 277, 923). Following a 1-weekacclimation, the mice receive a single tail vein injection ofoligonucleotide (5 mg/kg) administered in phosphate buffered saline(PBS), pH 7.0. The final concentration of oligonucleotide in the dosingsolution is (5 mg/kg) for the PBS formulations. One retro-orbital bleed(either 0.25, 9.05, 2 or 4 post dose) and a terminal bleed (either 1, 3,8 or 24 h post dose) is collected from each group. The terminal bleed(approximately 0.6-0.8 mL) is collected by cardiac puncture followingketamine/xylazine anesthesia. The blood is transferred to an EDTA-coatedcollection tube and centrifuged to obtain plasma. At termination, theliver and kidneys will be collected from each mouse. Plasma and tissueshomogenates will be used for analysis for determination of intactoligonucleotide content by CGE. All samples are immediately frozen ondry ice after collection and stored at −80C until analysis.

Procedure 9

RNase H Studies with Chimeric C3′-Endo and C2′-Endo ModifledOligonucleotides with and without Nuclease Resistant Caps

³²p Labeling of Oligonucleotides

The oligoribonucleotide (sense strand) was 5′-end labeled with 32P using[³²P]ATP, T4 polynucleotide kinase, and standard procedures (Ausubel, F.M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J.A., and Struhl, K., in Current Protocols in Molecular Biology, JohnWiley, New York (1989)). The labeled RNA was purified by electrophoresison 12% denaturing PAGE (Sambrook, J., Frisch, E. F., and Maniatis, T.,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor LaboratoryPress, Plainview (1989)). The specific activity of the labeledoligonucleotide was approximately 6000 cpm/fmol.

Determination of RNase H Cleavage Patterns

Hybridization reactions were prepared in 120 μL of reaction buffer [20mM Tris-HC (pH 7.5), 20 mM KCl, 10 mM MgCl₂, 0.1 mM DTT] containing 750nM antisense oligonucleotide, 500 nM sense oligoribonucleotide, and100,000 cpm ³²P-labeled sense oligoribonucleotide. Reactions were heatedat 90° C. for 5 min and 1 unit of Inhibit-ACE was added. Samples wereincubated overnight at 37° C. degrees. Hybridization reactions wereincubated at 37° C. with 1.5×10.8⁻⁸ mg of E. coli RNase H enzyme forinitial rate determinations and then quenched at specific time points.Samples were analyzed by trichloroacetic acid (TCA) assay or bydenaturing polyacrylamide gel electrophoresis as previously described[Crooke, S. T., Lemonidis, K. M., Neilson, L., Griffey, R., Lesnik, E.A., and Monia, B. P., Kinetic characteristics of Escherichia coli RNaseH1: cleavage of various antisense oligonucleotide-RNA duplexes, BiochemJ., 312, 599 (1995); Lima, W. F. and Crooke, S. T., Biochemistry 36,390-398, 1997].

Those skilled in the art will appreciate that numerous changes andmodifications can be made to the preferred embodiments of the inventionand that such changes and modifications can be made without departingfrom the spirit of the invention. It is therefore intended that theappended claims cover all such equivalent variations as fall within thetrue spirit and scope of the invention.

1. A mixed sequence oligonucleotide comprising at least 12 nucleotidesin length and having a 3′ end and a 5′ end and divided into a firstportion and a further portion, said first portion comprising nucleotidesthat support cleavage of a complementary target RNA by human RNase H1polypeptide, said further portion comprising nucleotides that do notsupport said cleavage by said RNase H1, wherein said first portioncomprises at least 6 contiguous nucleotides and is positioned in saidoligonucleotide such that at least one of said at least 6 contiguousnucleotides is 8 to 12 nucleotides from the 3′ end of saidoligonucleotide.
 2. A mixed sequence oligonucleotide comprising at least12 nucleotides and having a 3′ end and a 5′ end and divided into a firstportion and a further portion, said first portion supports cleavage of acomplementary target RNA by a purified or isolated human RNase H1polypeptide, said further portion does not support said cleavage by saidpurified or isolated RNase H1 and where said first portion comprises atleast 6 contiguous nucleotides and is positioned in said oligonucleotidesuch that at least one of said at least 6 contiguous nucleotides is 8 to12 nucleotides from the 3′ end of said oligonucleotide.
 3. Theoligonucleotide of claim 2 comprising from about 12 to about 50nucleotides.
 4. The oligonucleotide of claim 2 comprising from about 12to about 25 nucleotides.
 5. A mixed sequence oligonucleotide comprisingat least 12 nucleotides and having a 3′ end and a 5′ end and dividedinto a first portion and a further portion, said first portion supportscleavage of a complementary target RNA by human RNase H1 polypeptide,said further portion does not support said cleavage by said RNase H1;wherein: said first portion comprises at least 6 nucleotides and ispositioned in said oligonucleotide such that at least one of said atleast 6 nucleotides is 8 to 12 nucleotides from the 3′ end of saidoligonucleotide; said nucleotides of said first portion have B-formconformational geometry and are joined together in a continuoussequence, and at least one of said nucleotides having B-formconformational geometry is not a 2′-deoxyribonucleotide.
 6. A mixedsequence oligonucleotide comprising at least 12 nucleotides and having a3′ end and a 5′ end and divided into a first portion and a furtherportion, said first portion supports cleavage of a complementary targetRNA by human RNase H1 polypeptide, said further portion does not supportsaid cleavage by said RNase H1; wherein: said first portion comprises atleast 6 contiguous nucleotides and is positioned in said oligonucleotidesuch that at least one of said at least 6 contiguous nucleotides is 8 to12 nucleotides from the 3′ end of said oligonucleotide; and each of saidnucleotides of said first portion is, independently, a 2′-SCH₃ribonucleotide, a 2′-NH₂ ribonucleotide, a 2′-NH(C₁-C₂ alkyl)ribonucleotide, a 2′-N(C₁-C₂ alkyl)₂ ribonucleotide, a 2′-CF₃ribonucleotide, a 2′=CH₂ ribonucleotide, a 2′=CHF ribonucleotide, a2′=CF₂ ribonucleotide, a 2′-CH₃ ribonucleotide, a 2′-C₂H₅ribonucleotide, a 2′-CH═CH₂ ribonucleotide or a 2′-CCH ribonucleotide.7. The oligonucleotide of claim 2 wherein at least one of saidnucleotides of said first portion is a 2′-deoxyribonucleotide.
 8. Theoligonucleotide of claim 2 wherein said nucleotides of said firstportion are joined together in said continuous sequence byphosphorothioate linkages.
 9. The oligonucleotide of claim 2 whereinsaid further portion includes a plurality of nucleotides, at least someof said nucleotides comprise a 2′ substituent group wherein eachsubstituent group is, independently, hydroxyl, C₁-C₂₀ alkyl, C₂-C₂₀alkenyl, C₂-C₂₀ alkynyl, halogen, amino, thiol, keto, carboxyl, nitro,nitroso, nitrile, trifluoromethyl, trifluoromethoxy, O-alkyl, O-alkenyl,O-alkynyl, S-alkyl, S-alkenyl, S-alkynyl, NH-alkyl, NH-alkenyl,NH-alkynyl, N-dialkyl, O-aryl, S-aryl, NH-aryl, O-aralkyl, S-aralkyl,NH-aralkyl, N-phthalimido, imidazole, azido, hydrazino, hydroxylamino,isocyanato, sulfoxide, sulfone, sulfide, disulfide, silyl, aryl,heterocycle, carbocycle, intercalator, reporter molecule, conjugate,polyamine, polyamide, polyalkylene glycol, or polyether; or eachsubstituent group has one of formula I or II:

wherein: Z₀ is O, S or NH; J is a single bond, O or C(═O); E is C₁-C₁₀alkyl, N(R₁)(R₂), N═C(R₁)(R₂), or has one of formula III or IV;

each R₆, R₇, R₈, R₉ and R₁₀ is, independently, hydrogen, C(O)R₁₁,substituted or unsubstituted C₁-C₁₀ alkyl, substituted or unsubstitutedC₂-C₁₀ alkenyl, substituted or unsubstituted C₂-C₁₀ alkynyl,alkylsulfonyl, arylsulfonyl, a chemical functional group or a conjugategroup, wherein the substituent groups are selected from hydroxyl, amino,alkoxy, carboxy, benzyl, phenyl, nitro, thiol, thioalkoxy, halogen,alkyl, aryl, alkenyl and alkynyl; or optionally, R₇ and R₈, togetherform a phthalimido moiety with the nitrogen atom to which they areattached; or optionally, R₉ and R₁₀, together form a phthalimido moietywith the nitrogen atom to which they are attached; each R₁₁ is,independently, substituted or unsubstituted C₁-C₁₀ alkyl,trifluoromethyl, cyanoethyloxy, methoxy, ethoxy, t-butoxy, allyloxy,9-fluorenylmethoxy, 2-(trimethylsilyl)-ethoxy, 2,2,2-trichloroethoxy,benzyloxy, butyryl, iso-butyryl, phenyl or aryl; each R₁ and R₂ is,independently, H, a nitrogen protecting group, substituted orunsubstituted C₁-C₁₀ alkyl, substituted or unsubstituted C₂-C₁₀ alkenyl,substituted or unsubstituted C₂-C₁₀ alkynyl, wherein said substitutionis OR₃, SR₃, NH₃ ⁺, N(R₃)(R₄), guanidino or acyl where said acyl is anacid amide or an ester; or R₁ and R₂, together, are a nitrogenprotecting group or are joined in a ring structure that optionallyincludes an additional heteroatom selected from N and O; or R₁, T and L,together, are a chemical functional group; each R₃ and R₄ is,independently, H, C₁-C₁₀ alkyl, a nitrogen protecting group, or R₃ andR₄, together, are a nitrogen protecting group; or R₃ and R₄ are joinedin a ring structure that optionally includes an additional heteroatomselected from N and O; Z₄ is OX, SX, or N(X)₂; each X is, independently,H, C₁-C₈ alkyl, C₁-C₈ haloalkyl, C(═NH)N(H)R₅, C(═O)N(H)R₅ orOC(═O)N(H)R₅; R₅ is H or C₁-C₈ alkyl; Z₁, Z₂ and Z₃ comprise a ringsystem having from about 4 to about 7 carbon atoms or having from about3 to about 6 carbon atoms and 1 or 2 hetero atoms wherein said heteroatoms are selected from oxygen, nitrogen and sulfur and wherein saidring system is aliphatic, unsaturated aliphatic, aromatic, or saturatedor unsaturated heterocyclic; Z₅ is alkyl or haloalkyl having 1 to about10 carbon atoms, alkenyl having 2 to about 10 carbon atoms, alkynylhaving 2 to about 10 carbon atoms, aryl having 6 to about 14 carbonatoms, N(R₁)(R₂) OR, halo, SR₁ or CN; each q₁ is, independently, aninteger from 1 to 10; each q₂ is, independently, 0 or 1; q₃ is 0 or aninteger from 1 to 10; q₄ is an integer from 1 to 10; and q₅ is 0, 1 or2; provided that when q₃ is 0, q₄ is greater than
 1. 10. Theoligonucleotide of claim 2 wherein said further portion comprises aplurality of nucleotides and wherein each of said nucleotides of saidfurther portion is, independently, a 2′-F ribonucleotide, a 2′-O-(C₁-C₆alkyl) ribonucleotide, or a 2′-O-(C₁-C₆ substituted alkyl)ribonucleotide wherein the substitution is C₁-C₆ ether, C₁-C₆ thioether,amino, amino(C₁-C₆ alkyl) or amino(C₁-C₆ alkyl)₂.
 11. Theoligonucleotide of claim 2 wherein said further portion comprises aplurality of nucleotides and wherein at least two of said nucleotides ofsaid further portion are joined together in a continuous sequence thatis positioned 3′ to said first portion.
 12. The oligonucleotide of claim2 wherein said further portion comprises a plurality of nucleotides andwherein at least two of said nucleotides of said further portion arejoined together in a continuous sequence that is positioned 5′ to saidfirst portion.
 13. The oligonucleotide of claim 2 wherein said furtherportion comprises a plurality of nucleotides and wherein at least two ofsaid nucleotides of said further portion are joined together in acontinuous sequence that is positioned 3′ to said first portion and atleast two of said further portion are joined together in a continuoussequence that is positioned 5′ to said first portion.
 14. Theoligonucleotide of claim 2 wherein said further portion comprises aplurality of nucleotides and wherein at least four of said nucleotidesof said further portion are joined together in a continuous sequencethat is positioned 3′ to said first portion.
 15. The oligonucleotide ofclaim 2 wherein said further portion comprises a plurality ofnucleotides and wherein at least four of said nucleotides of saidfurther portion are joined together in a continuous sequence that ispositioned 5′ to said first portion.
 16. The oligonucleotide of claim 2wherein said further portion comprises a plurality of nucleotides andwherein at least four of said nucleotides of said further portion arejoined together in a continuous sequence that is positioned 3′ to saidfirst portion and at least four of said nucleotides of said furtherportion are joined together in a continuous sequence that is positioned5′ to said first portion.
 17. A mixed sequence oligonucleotidecomprising at least 8 oligonucleotides and having a 2′-OHarabinonucleotide sequence of at least 6 nucleotides and where at leastone of said arabinonucleotides is positioned 8 to 12 nucleotides fromthe 3′ end of said oligonucleotide, and wherein said oligonucleotidesupports cleavage of a complementary target RNA by human RNase H1polypeptide.
 18. A mixed sequence oligonucleotide comprising at least 8oligonucleotides and having a a 2′-F arabinonucleotide sequence of atleast 6 nucleotides and where at least one of said 2′-Farabinonucleotides is positioned 8 to 12 nucleotides from the 3′ end ofsaid oligonucleotide, and wherein said oligonucleotide supports cleavageof a complementary target RNA by human RNase H1 polypeptide.
 19. A mixedsequence oligonucleotide comprising 8 to 25 nucleotides and having a2′-OH arabinonucleotide sequence wherein at least one of the nucleotidesof said sequence is positioned 8 to 12 nucleotides from the 3′ end ofsaid oligonucleotide, wherein said oligonucleotide supports cleavage ofa complementary target RNA by human RNase H1 polypeptide.
 20. A mixedsequence oligonucleotide comprising 8 to 25 nucleotides and having a2′-F arabinonucleotide sequence wherein at least one of the nucleotidesof said sequence is positioned 8 to 12 nucleotides from the 3′ end ofsaid oligonucleotide, wherein said oligonucleotide supports cleavage ofa complementary target RNA by human RNase H1 polypeptide.
 21. A chimericoligonucleotide comprising 8 to 25 nucleotides and having a portion thatsupports cleavage of a complementary target RNA by human RNase H1polypeptide wherein said portion supporting said cleavage is at least 6contiguous nucleotides in length and is positioned in saidoligonucleotide such that at least one of said at least 6 contiguousnucleotides is positioned 8 to 12 nucleotides from the 3′ end of saidoligonucleotide, wherein said oligonucleotide supports cleavage of acomplementary target RNA by human RNase H1 polypeptide.
 22. Theoligonucleotide of claim 21 wherein said RNase H1 polypeptide is apurified or isolated polypeptide.
 23. A method comprising contacting anoligonucleotide according to claim 2 with RNA or DNA in vitro.
 24. Amethod comprising contacting an oligonucleotide according to claim 5with RNA or DNA in vitro.
 25. A method comprising contacting anoligonucleotide according to claim 6 with RNA or DNA in vitro.
 26. Amethod comprising contacting an oligonucleotide according to claim 17with RNA or DNA in vitro.
 27. A method comprising contacting anoligonucleotide according to claim 18 with RNA or DNA in vitro.
 28. Amethod comprising contacting an oligonucleotide according to claim 19with RNA or DNA in vitro.
 29. A method comprising contacting anoligonucleotide according to claim 20 with RNA or DNA in vitro.
 30. Amethod comprising contacting an oligonucleotide according to claim 2with RNA or DNA in a cellular assay.
 31. A method comprising contactingan oligonucleotide according to claim 5 with RNA or DNA in a cellularassay.
 32. A method comprising contacting an oligonucleotide accordingto claim 6 with RNA or DNA in a cellular assay.
 33. A method comprisingcontacting an oligonucleotide according to claim 17 with RNA or DNA in acellular assay.
 34. A method comprising contacting an oligonucleotideaccording to claim 18 with RNA or DNA in a cellular assay.
 35. A methodcomprising contacting an oligonucleotide according to claim 19 with RNAor DNA in a cellular assay.
 36. A method comprising contacting anoligonucleotide according to claim 20 with RNA or DNA in a cellularassay.
 37. A method comprising contacting an oligonucleotide accordingto claim 21 with RNA or DNA in vitro.
 38. A method comprising contactingan oligonucleotide according to claim 21 with RNA or DNA in a cellularassay.
 39. A method comprising contacting an oligonucleotide accordingto claim 22 with RNA or DNA in vitro.
 40. A method comprising contactingan oligonucleotide according to claim 22 with RNA or DNA in a cellularassay.
 41. A method comprising: selecting a parameter associated withinteraction of an oligonucleotide, a target nucleic acid and human RNaseH1 polypeptide, selecting a cell containing human RNase H1 polypeptideand a target nucleic acid, selecting first and second oligonucleotideseach having a sequence substantially commpementary to said targetnucleic acid, contacting said cell with said first oligonucleotide andmeasuring said parameter, contacting said cell with said secondoligonucleotide and measuring said parameter, comparing measurements,and using said comparison to select one of said first or said secondoligonucleotides for modulating said target in the presence of saidhuman RNase H1 polypeptide in said cell.
 42. The method of claim 41wherein said parameter is one of site preference for cleavage, sequencepreference for cleavage or processivity of cleavage.
 43. The method ofclaim 41 wherein said measurement is a measurement of at least one ofK_(d), K_(max), K_(m) or K_(cat).
 44. An oligonucleotide for modulatinga target nucleic acid in the presence of human RNase H1 polypeptidewherein said oligonucleotide is identified using the process of claim41.
 45. An optimized oligonucleotide for modulating a target nucleicacid wherein said optimized oligonucleotide is identified using theprocess of claim
 41. 46. The method of claim 41 further comprising:selecting a further oligonucleotide, contacting said cell with saidfurther oligonucleotide and measuring said parameter, comparing themeasurement of this further oligonucleotide to that of said selectedfirst or said second oligonucleotides and selecting one of said first,said second or said further oligonucleotide.